Fc variants with altered binding to fcrn

ABSTRACT

The present application relates to a variant Fc region comprising the double mutation 428L/434S that increases serum half-life and the numbering is according to the EU index.

This application is a continuation of U.S. patent application Ser. No.12/341,769, filed on Dec. 22, 2008, U.S. Ser. No. 12/341,769 claimsbenefit under 35 U.S.C. §119(e) to U.S. Ser. No. 61/016,793, filed Dec.26, 2007; U.S. Ser. No. 61/031,353, filed Feb. 25, 2008; U.S. Ser. No.61/046,353, filed Apr. 18, 2008; U.S. Ser. No. 61/050,172, filed May 2,2008; U.S. Ser. No. 61/079,779, filed Jul. 10, 2008; U.S. Ser. No.61/099,178, and filed Sep. 22, 2008; and U.S. Ser. No. 12/341,769 is acontinuation-in-part of U.S. Ser. No. 11/932,151, filed Oct. 31, 2007,U.S. Ser. No. 11/932,151 claims the benefit under 35 U.S.C. §119(e) toU.S. Ser. No. 60/951,536, filed Jul. 24, 2007, U.S. Ser. No. 12/341,769is a continuation-in-part of U.S. Ser. No. 11/436,266, filed May 17,2006, U.S. Ser. No. 11/436,266 is a continuation-in-part of U.S.application Ser. No. 11/274,065, filed Nov. 14, 2005; U.S. Ser. No.11/274,065 claims benefit under 35 U.S.C. §119(e) to U.S. Ser. No.60/627,763 filed Nov. 12, 2004, U.S. Ser. No. 60/642,886, filed Jan. 11,2005, U.S. Ser. No. 60/649,508, filed Feb. 2, 2005, U.S. Ser. No.60/662,468, filed Mar. 15, 2005, U.S. Ser. No. 60/669,311, filed Apr. 6,2005, U.S. Ser. No. 60/681,607, filed May 16, 2005, U.S. Ser. No.60/690,200, filed Jun. 13, 2005, U.S. Ser. No. 60/696,609, filed Jul. 5,2005, U.S. Ser. Nos. 60/703,018, filed Jul. 27, 2005, and 60/726,453,filed Oct. 12, 2005, all entirely incorporated by reference.

FIELD OF THE INVENTION

The present application relates to optimized IgG immunoglobulinvariants, engineering methods for their generation, and theirapplication, particularly for therapeutic purposes.

BACKGROUND OF THE INVENTION

Antibodies are immunological proteins that each binds a specificantigen. In most mammals, including humans and mice, antibodies areconstructed from paired heavy and light polypeptide chains. Each chainis made up of individual immunoglobulin (Ig) domains, and thus thegeneric term immunoglobulin is used for such proteins. Each chain ismade up of two distinct regions, referred to as the variable andconstant regions. The light and heavy chain variable regions showsignificant sequence diversity between antibodies, and are responsiblefor binding the target antigen. The constant regions show less sequencediversity, and are responsible for binding a number of natural proteinsto elicit important biochemical events. In humans there are fivedifferent classes of antibodies including IgA (which includes subclassesIgA1 and IgA2), IgD, IgE, IgG (which includes subclasses IgG1, IgG2,IgG3, and IgG4), and IgM. The distinguishing feature between theseantibody classes is their constant regions, although subtler differencesmay exist in the V region. IgG antibodies are tetrameric proteinscomposed of two heavy chains and two light chains. The IgG heavy chainis composed of four immunoglobulin domains linked from N- to C-terminusin the order VH-CH1-CH2-CH3, referring to the heavy chain variabledomain, heavy chain constant domain 1, heavy chain constant domain 2,and heavy chain constant domain 3 respectively (also referred to asVH-Cγ1-Cγ2-Cγ3, referring to the heavy chain variable domain, constantgamma 1 domain, constant gamma 2 domain, and constant gamma 3 domainrespectively). The IgG light chain is composed of two immunoglobulindomains linked from N- to C-terminus in the order VL-CL, referring tothe light chain variable domain and the light chain constant domainrespectively.

In IgG, a site on Fc between the Cγ2 and Cγ3 domains mediatesinteraction with the neonatal receptor FcRn. Binding to FcRn recyclesendocytosed antibody from the endosome back to the bloodstream (Raghavanet al., 1996, Annu Rev Cell Dev Biol 12:181-220; Ghetie et al., 2000,Annu Rev Immunol 18:739-766, both entirely incorporated by reference).This process, coupled with preclusion of kidney filtration due to thelarge size of the full-length molecule, results in favorable antibodyserum half-lives ranging from one to three weeks. Binding of Fc to FcRnalso plays a key role in antibody transport. The binding site on Fc forFcRn is also the site at which the bacterial proteins A and G bind. Thetight binding by these proteins is typically exploited as a means topurify antibodies by employing protein A or protein G affinitychromatography during protein purification. Thus the fidelity of thisregion on Fc is important for both the clinical properties of antibodiesand their purification. Available structures of the rat Fc/FcRn complex(Burmeister et al., 1994, Nature, 372:379-383; Martin et al., 2001, MolCell 7:867-877, both entirely incorporated by reference), and of thecomplexes of Fc with proteins A and G (Deisenhofer, 1981, Biochemistry20:2361-2370; Sauer-Eriksson et al., 1995, Structure 3:265-278; Tashiroet al., 1995, Curr Opin Struct Biol 5:471-481, all entirely incorporatedby reference), provide insight into the interaction of Fc with theseproteins. The FcRn receptor is also responsible for the transfer of IgGto the neo-natal gut and to the lumen of the intestinal epithelia inadults (Ghetie and Ward, Annu. Rev. Immunol., 2000, 18:739-766; Yoshidaet al., Immunity, 2004, 20(6):769-783, both entirely incorporated byreference).

Studies of rat and human Fc domains have demonstrated the importance ofsome Fc residues to the binding of FcRn. The rat and human sequenceshave about 64% sequence identity in the Fc regions (residues 237-443 inthe numbering of EU index). See FIGS. 3, 4, and 5 for the rat/humanalignments of Fc, FcRn heavy chain, and FcRn light chain(beta-2-microglobulin). A model of the human Fc/FcRn complex has beenbuilt from the existing structure of the rat Fc/FcRn complex (Martin etal., 2001, Mol Cell 7:867-877, entirely incorporated by reference). Therat and human sequences share some residues that are critical for FcRnbinding, such as H310 and H435 (Medesan et al., 1997 J. Immunol.158(5):221-7; Shields et al., 2001, J. Biol. Chem. 276(9):6591-6604,both entirely incorporated by reference). In many positions, however,the human and rat proteins have different amino acids, giving theresidues in the human sequence different environments, and possibly adifferent identities, than in the rat sequence. This variability limitsthe ability to transfer characteristics from one homolog to the otherhomolog.

In the murine Fc, random mutation and phage display selection at thesites, T252, T254, and T256 lead to a triple mutant, T252L/T254S/T256F,that has a 3.5-fold increase in FcRn affinity and a 1.5-fold increase inserum half-life (Ghetie et al., 1997, Nat. Biotech. 15(7): 637-640,entirely incorporated by reference). Disruption of the Fc/FcRninteraction by mutations at positions 253, 310 and 435 also lead todecreased in vivo half-life (Medesan et al J. Immunol. 1997158(5):2211-7, entirely incorporated by reference).

Mutational studies in human Fcγ have been done on some of the residuesthat are important for binding to FcRn and have demonstrated anincreased serum half-life. In human Fcγ1, Hinton et al. mutated threeresidues individually to the other 19 common amino acids. Hinton et al.,found that some point mutants a double mutant increased the FcRn bindingaffinity (Hinton et al., 2004, J. Biol. Chem. 279(8): 6213-6216; Hintonet al. Journal of Immunology 2006, 176:346-356, both entirelyincorporated by reference). Two mutations had increased half-lives inmonkeys. Shields et al. mutated residues, almost exclusively to Ala, andstudied their binding to FcRn and the FcγR's (Shields et al., 2001, J.Biol. Chem., 276(9):6591-6604, entirely incorporated by reference).

Dall'Acqua et al. used phage display to select for Fc mutations thatbound FcRn with increased affinity (Dall' Acqua et al. 2002, J. Immunol.169:5171-5180, entirely incorporated by reference). The DNA sequencesselected for were primarily double and triple mutants. The referenceexpressed the proteins encoded by many of their selected sequences andfound some that bound to FcRn more tightly than the wild-type Fc.

The administration of antibodies and Fc fusion proteins as therapeuticsrequires injections with a prescribed frequency relating to theclearance and half-life characteristics of the protein. Longer in vivohalf-lives allow more seldom injections or lower dosing, which isclearly advantageous. Although the past mutations in the Fc domain havelead to some proteins with increased FcRn binding affinity and in vivohalf-lives, these mutations have not identified the optimal mutationsand enhanced in vivo half-life.

One feature of the Fc region is the conserved N-linked glycosylationthat occurs at N297. This carbohydrate, or oligosaccharide as it issometimes called, plays a critical structural and functional role forthe antibody, and is one of the principle reasons that antibodies mustbe produced using mammalian expression systems. Umaña et al., 1999, NatBiotechnol 17:176-180; Davies et al., 2001, Biotechnol Bioeng74:288-294; Mimura et al., 2001, J Biol Chem 276:45539-45547.; Radaev etal., 2001, J Biol Chem 276:16478-16483; Shields et al., 2001, J BiolChem 276:6591-6604; Shields et al., 2002, J Biol Chem 277:26733-26740;Simmons et al., 2002, J Immunol Methods 263:133-147; Radaev et al.,2001, J Biol Chem 276:16469-16477; and Krapp et al., 2003, J Mol Biol325:979-989, all entirely incorporated by reference).

Antibodies have been developed for therapeutic use. Representativepublications related to such therapies include Chamow et al., 1996,Trends Biotechnol 14:52-60; Ashkenazi et al., 1997, Curr Opin Immunol9:195-200, Cragg et al., 1999, Curr Opin Immunol 11:541-547; Glennie etal., 2000, Immunol Today 21:403-410, McLaughlin et al., 1998, J ClinOncol 16:2825-2833, and Cobleigh et al., 1999, J Clin Oncol17:2639-2648, all entirely incorporated by reference. Currently foranticancer therapy, any small improvement in mortality rate definessuccess. Certain IgG variants disclosed herein enhance the capacity ofantibodies to limit further growth or destroy at least partially,targeted cancer cells.

Anti-tumor potency of antibodies is via enhancement of their ability tomediate cytotoxic effector functions such as ADCC, ADCP, and CDC.Examples include Clynes et al., 1998, Proc Nat/Acad Sci USA 95:652-656;Clynes et al., 2000, Nat Med 6:443-446 and Cartron et al., 2002, Blood99:754-758, both entirely incorporated by reference.

Human IgG1 is the most commonly used antibody for therapeutic purposes,and the majority of engineering studies have been constructed in thiscontext. The different isotypes of the IgG class however, includingIgG1, IgG2, IgG3, and IgG4, have unique physical, biological, andclinical properties. There is a need in the art to design improved IgG1,IgG2, IgG3, and IgG4 variants. There is a further need to design suchvariants to improve binding to FcRn and/or increase in vivo half-life ascompared to native IgG polypeptides. Additionally, there is a need tocombine variants with improved pharmacokinetic properties with variantscomprising modifications to improve efficacy through altered FcgammaRbinding. The present application meets these and other needs.

SUMMARY OF THE INVENTION

The present application is directed to Fc variants of a parentpolypeptide including at least one modification in the Fc region of thepolypeptide. In various embodiments, the variant polypeptides exhibitaltered binding to FcRn as compared to a parent polypeptide. In certainvariations, the modification can be selected from the group consistingof: 428L, 434M and 434S, where the numbering is according to the EUIndex in Kabat et al.

In another embodiment, the Fc variant includes at least twomodifications selected from the group consisting of: 252Y/428L,428L/434H, 428L/434F, 428L/434Y, 428L/434A, 428L/434M, and 428L/434S.

In another embodiment, the Fc variant includes at least one modificationselected from the group consisting of: M428L/N434S, V308F/M428L/N434S.

In another embodiment, the Fc variant includes at least one modificationselected from the group consisting of: 259I/434S, 308F/434S,308F/428L/434S, 259I/308F/434S, 307Q/308F/434S, 250I/308F/434S, and308F/319L/434S.

In another embodiment, the Fc variant includes at least one modificationselected from the group consisting of:

In another embodiment, the invention includes a method of treating apatient in need of said treatment comprising administering an effectiveamount of an Fc variant described herein.

In another embodiment, the invention includes a method of increasing thehalf-life of an antibody or immunoadhesin by modifying an Fc accordingto the modifications described herein.

In another variant, the invention includes Fc variant with enchancedFcRn binding with additional Fc variants that modulate effectorfunction.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Sequence alignments of human IgG constant heavy chains. Grayindicates differences from IgG1, and boxed residues indicate commonallotypic variations in the human population.

FIG. 2. (SEQ ID NO: 1-6) Amino acid sequences of constant regions usedin the invention.

FIG. 3. (SEQ ID NO: 7-12) Amino acid sequences of exemplary variantconstant regions.

FIG. 4. (SEQ ID NO: 13-22) Amino acid sequences of VH and VL variableregions used in the invention.

FIG. 5. (SEQ ID NO: 23-29) Amino acid sequences of exemplary variantantibodies.

FIG. 6. Relative VEGF binding by WT and select variant IgG1 anti-VEGFantibodies. The plot shows the Biacore response units (RU) at the end ofthe association phase for binding of antibody analyte to immobilizedVEGF antigen. Anti-Her2 IgG1 antibody was used as a negative control.

FIG. 7. Biacore sensorgrams of WT and variant IgG1 antibodies toimmobilized human FcRn at low (6.0) and high (7.4) pH.

FIG. 8. FcRn binding affinities of WT and select variant IgG1 antibodiesto human FcRn at pH 6.0 as determined by Biacore. The graph shows a plotof the pseudo-affinity constant (Ka*), on a log scale.

FIG. 9. Relative binding of variant IgG1 anti-VEGF antibodies to humanFcRn as determined by Biacore. The table shows the fold of the Ka* ofeach variant relative to human WT (native) IgG1. n indicates the numberof time each variant was tested, and Mean and SD indicate the averageand standard deviation respectively for each variant over n bindingexperiments. Fold FcRn was calculated for all variants relative to WTIgG1 within each respective binding experiment. NB indicates no bindingwas detected. ND indicates that binding was not determined for thatparticular variant. NF indicates no fit was possible from the bindingdata.

FIG. 10. Relative binding of variant IgG2 and IgG1/2 anti-VEGFantibodies to human FcRn as determined by Biacore. The table is asdescribed in FIG. 9.

FIG. 11. Analysis of additive and synergistic substitution combinations.FIG. 11 a shows a plot of the experimentally determined fold binding tohuman FcRn by each variant versus the predicted fold FcRn binding asdetermined by the product of the single variants. Variant data pointsare labeled, and the line represents perfect additivity. FIG. 11 b showsthe difference between experimental and predicted fold for eachcombination variant. FIG. 11 c shows the synergy of each variantcombination. % synergy is calculated as the 100×[(experimentalfold/predicted fold)−1)].

FIG. 12. Relative binding of variant anti-TNF, -CD25, -EGFR, and -IgEantibodies to human FcRn as determined by Biacore. The table is asdescribed in FIG. 9.

FIG. 13. In vivo pharmacokinetics of WT and variant antibodies inmFcRn−/− hFcRn+ mice. The graphs plot the serum concentration ofantibody versus time after a single intravenous dose. FIG. 13 a showsdata from one of the 4 studies carried out with IgG1 antibodies (Study3), and FIG. 13 b shows data from a study carried out with IgG2antibodies (Study 5).

FIG. 14. Fitted PK parameters from all in vivo PK studies carried out inmFcRn−/− hFcRn+ mice with variant and WT antibodies. n represents thenumber of mice per group, with Mean and standard deviation (SD) dataprovided for PK parameters. Half-Life represents the beta phase thatcharacterizes elimination of antibody from serum. Cmax is the maximalobserved serum concentration, AUC is the area under the concentrationtime curve, and clearance is the clearance of antibody from serum. Foldhalf-life is calculated as the half-life of variant antibody over thatof the WT IgG1 or IgG2 parent within each study.

FIG. 15. Correlation between half-life of IgG1 (FIG. 15 a) and IgG2(FIG. 15 b) variant antibodies in mFcRn−/− hFcRn+ mice and fold FcRnbinding relative to WT IgG1. Data on the y-axis are from FIG. 14, anddata on the x-axis are from FIGS. 9 and 10. Select variants are labeled,and variant data from repeat experiments are circled. FIG. 15 c showsboth IgG1 and IgG2 correlation data, with the black and gray linesrepresenting fits of the IgG1 and IgG2 data respectively.

FIG. 16. (SEQ ID NO: 30-35) Amino acid sequences of variant and parentanti-TNF Fc immunoadhesins used in the invention.

FIG. 17. Binding of anti-TNF immunoadhesins to TNF antigen as determinedby Biacore.

FIG. 18. Relative binding of variant Fc immunoadhesins to human FcRn asdetermined by Biacore. The table shows the fold of the Ka* of eachvariant relative to human WT (native) IgG1. n indicates the number oftime each variant was tested, and Mean and SD indicate the average andstandard deviation respectively for each variant over n bindingexperiments. Fold FcRn was calculated for all variants relative to therespective IgG parent within each respective binding experiment.

FIG. 19. In vivo pharmacokinetics of parent and variant Fcimmunoadhesins in mFcRn−/− hFcRn+ mice. The graphs plot the serumconcentration of Fc fusion versus time after a single intravenous dose.

FIG. 20. Fitted PK parameters from the Fc fusion in vivo PK study inmFcRn−/− hFcRn+ mice. Parameters are as described in FIG. 14. % increasein half-life is calculated as 100 times the half-life of variant Fcfusion over that of the WT IgG1 or IgG2 parent.

FIG. 21. Relative binding of variant IgG1 anti-VEGF antibodies tocynomolgus monkey and human FcRn as determined by Biacore. FIG. 21 ashows the data in tabular form. Description of the figure is as in FIG.9, and data for binding to human FcRn are taken from FIG. 9. FIG. 21 bshows a plot of the data.

FIG. 22. In vivo pharmacokinetics of WT and variant antibodies incynomolgus monkeys. The graphs plot the serum concentration of antibodyversus time after a single intravenous dose.

FIG. 23. Fitted PK parameters from the in vivo PK study in cynomolgusmonkeys with variant and WT antibodies. Parameters are as described inFIG. 14.

DETAILED DESCRIPTION OF THE INVENTION

The present invention discloses the generation of novel variants of Fcdomains, including those found in antibodies, Fc fusions, andimmuno-adhesions, which have an increased binding to the FcRn receptor.As noted herein, binding to FcRn results in longer serum retention invivo.

In order to increase the retention of the Fc proteins in vivo, theincrease in binding affinity must be at around pH 6 while maintaininglower affinity at around pH 7.4. Although still under examination, Fcregions are believed to have longer half-lives in vivo, because bindingto FcRn at pH 6 in an endosome sequesters the Fc (Ghetie and Ward, 1997Immunol Today. 18(12): 592-598, entirely incorporated by reference). Theendosomal compartment then recycles the Fc to the cell surface. Once thecompartment opens to the extracellular space, the higher pH, ˜7.4,induces the release of Fc back into the blood. In mice, Dall' Acqua etal. showed that Fc mutants with increased FcRn binding at pH 6 and pH7.4 actually had reduced serum concentrations and the same half life aswild-type Fc (Dall'Acqua et al. 2002, J. Immunol. 169:5171-5180,entirely incorporated by reference). The increased affinity of Fc forFcRn at pH 7.4 is thought to forbid the release of the Fc back into theblood. Therefore, the Fc mutations that will increase Fc's half-life invivo will ideally increase FcRn binding at the lower pH while stillallowing release of Fc at higher pH. The amino acid histidine changesits charge state in the pH range of 6.0 to 7.4. Therefore, it is notsurprising to find His residues at important positions in the Fc/FcRncomplex (FIG. 6.)

An additional aspect of the invention is the increase in FcRn bindingover wild type specifically at lower pH, about pH 6.0, to facilitateFc/FcRn binding in the endosome. Also disclosed are Fc variants withaltered FcRn binding and altered binding to another class of Fcreceptors, the FcγR's (sometimes written FcgammaR's) as differentialbinding to FcγRs, particularly increased binding to FcγRIIIb anddecreased binding to FcγRIIb, has been shown to result in increasedefficacy.

DEFINITIONS

In order that the application may be more completely understood, severaldefinitions are set forth below. Such definitions are meant to encompassgrammatical equivalents.

By “ADCC” or “antibody dependent cell-mediated cytotoxicity” as usedherein is meant the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause lysis of the target cell.

By “ADCP” or antibody dependent cell-mediated phagocytosis as usedherein is meant the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause phagocytosis of the target cell.

By “modification” herein is meant an amino acid substitution, insertion,and/or deletion in a polypeptide sequence or an alteration to a moietychemically linked to a protein. For example, a modification may be analtered carbohydrate or PEG structure attached to a protein. By “aminoacid modification” herein is meant an amino acid substitution,insertion, and/or deletion in a polypeptide sequence.

By “amino acid substitution” or “substitution” herein is meant thereplacement of an amino acid at a particular position in a parentpolypeptide sequence with another amino acid. For example, thesubstitution E272Y refers to a variant polypeptide, in this case an Fcvariant, in which the glutamic acid at position 272 is replaced withtyrosine.

By “amino acid insertion” or “insertion” as used herein is meant theaddition of an amino acid sequence at a particular position in a parentpolypeptide sequence. For example, -233E or ̂233E designates aninsertion of glutamic acid after position 233 and before position 234.Additionally, -233ADE or ̂233ADE designates an insertion of AlaAspGluafter position 233 and before position 234.

By “amino acid deletion” or “deletion” as used herein is meant theremoval of an amino acid sequence at a particular position in a parentpolypeptide sequence. For example, E233- or E233# designates a deletionof glutamic acid at position 233. Additionally, EDA233- or EDA233#designates a deletion of the sequence GluAspAla that begins at position233.

By “variant protein” or “protein variant”, or “variant” as used hereinis meant a protein that differs from that of a parent protein by virtueof at least one amino acid modification. Protein variant may refer tothe protein itself, a composition comprising the protein, or the aminosequence that encodes it. Preferably, the protein variant has at leastone amino acid modification compared to the parent protein, e.g. fromabout one to about seventy amino acid modifications, and preferably fromabout one to about five amino acid modifications compared to the parent.The protein variant sequence herein will preferably possess at leastabout 80% homology with a parent protein sequence, and most preferablyat least about 90% homology, more preferably at least about 95%homology. Variant protein can refer to the variant protein itself,compositions comprising the protein variant, or the DNA sequence thatencodes it. Accordingly, by “antibody variant” or “variant antibody” asused herein is meant an antibody that differs from a parent antibody byvirtue of at least one amino acid modification, “IgG variant” or“variant IgG” as used herein is meant an antibody that differs from aparent IgG by virtue of at least one amino acid modification, and“immunoglobulin variant” or “variant immunoglobulin” as used herein ismeant an immunoglobulin sequence that differs from that of a parentimmunoglobulin sequence by virtue of at least one amino acidmodification. “Fc variant” or “variant Fc” as used herein is meant aprotein comprising a modification in an Fc domain. The Fc variants ofthe present invention are defined according to the amino acidmodifications that compose them. Thus, for example, N434S or 434S is anFc variant with the substitution serine at position 434 relative to theparent Fc polypeptide, wherein the numbering is according to the EUindex. Likewise, M428L/N434S defines an Fc variant with thesubstitutions M428L and N434S. A relative to the parent Fc polypeptide.The identity of the WT amino acid may be unspecified, in which case theaforementioned variant is referred to as 428L/434S. It is noted that theorder in which substitutions are provided is arbitrary, that is to saythat, for example, 428L/434S is the same Fc variant as M428L/N434S, andso on. For all positions discussed in the present invention, numberingis according to the EU index. The EU index or EU index as in Kabat or EUnumbering scheme refers to the numbering of the EU antibody (Edelman etal., 1969, Proc Natl Acad Sci USA 63:78-85, hereby entirely incorporatedby reference.) The modification can be an addition, deletion, orsubstitution. Substitutions can include naturally occurring amino acidsand non-naturally occurring amino acids. Variants may comprisenon-natural amino acids. Examples include U.S. Pat. No. 6,586,207; WO98/48032; WO 03/073238; US2004-0214988A1; WO 05/35727A2; WO 05/74524A2;J. W. Chin et al., (2002), Journal of the American Chemical Society124:9026-9027; J. W. Chin, & P. G. Schultz, (2002), ChemBioChem11:1135-1137; J. W. Chin, et al., (2002), PICAS United States of America99:11020-11024; and, L. Wang, & P. G. Schultz, (2002), Chem. 1-10, allentirely incorporated by reference.

As used herein, “protein” herein is meant at least two covalentlyattached amino acids, which includes proteins, polypeptides,oligopeptides and peptides. The peptidyl group may comprise naturallyoccurring amino acids and peptide bonds, or synthetic peptidomimeticstructures, i.e. “analogs”, such as peptoids (see Simon et al., PNAS USA89(20):9367 (1992), entirely incorporated by reference). The amino acidsmay either be naturally occurring or non-naturally occurring; as will beappreciated by those in the art. For example, homo-phenylalanine,citrulline, and noreleucine are considered amino acids for the purposesof the invention, and both D- and L- (R or S) configured amino acids maybe utilized. The variants of the present invention may comprisemodifications that include the use of unnatural amino acids incorporatedusing, for example, the technologies developed by Schultz andcolleagues, including but not limited to methods described by Cropp &Shultz, 2004, Trends Genet. 20(12):625-30, Anderson et al., 2004, ProcNatl Acad Sci USA 101(2):7566-71, Zhang et al., 2003, 303(5656):371-3,and Chin et al., 2003, Science 301(5635):964-7, all entirelyincorporated by reference. In addition, polypeptides may includesynthetic derivatization of one or more side chains or termini,glycosylation, PEGylation, circular permutation, cyclization, linkers toother molecules, fusion to proteins or protein domains, and addition ofpeptide tags or labels.

By “residue” as used herein is meant a position in a protein and itsassociated amino acid identity. For example, Asparagine 297 (alsoreferred to as Asn297 or N297) is a residue at position 297 in the humanantibody IgG1.

By “Fab” or “Fab region” as used herein is meant the polypeptide thatcomprises the VH, CH1, VL, and CL immunoglobulin domains. Fab may referto this region in isolation, or this region in the context of a fulllength antibody, antibody fragment or Fab fusion protein. By “Fv” or “Fvfragment” or “Fv region” as used herein is meant a polypeptide thatcomprises the VL and VH domains of a single antibody.

By “IgG subclass modification” as used herein is meant an amino acidmodification that converts one amino acid of one IgG isotype to thecorresponding amino acid in a different, aligned IgG isotype. Forexample, because IgG1 comprises a tyrosine and IgG2 a phenylalanine atEU position 296, a F296Y substitution in IgG2 is considered an IgGsubclass modification.

By “non-naturally occurring modification” as used herein is meant anamino acid modification that is not isotypic. For example, because noneof the IgGs comprise a serine at position 434, the substitution 434S inIgG1, IgG2, IgG3, or IgG4 is considered a non-naturally occurringmodification.

By “amino acid” and “amino acid identity” as used herein is meant one ofthe 20 naturally occurring amino acids or any non-natural analogues thatmay be present at a specific, defined position.

By “effector function” as used herein is meant a biochemical event thatresults from the interaction of an antibody Fc region with an Fcreceptor or ligand. Effector functions include but are not limited toADCC, ADCP, and CDC.

By “IgG Fc ligand” as used herein is meant a molecule, preferably apolypeptide, from any organism that binds to the Fc region of an IgGantibody to form an Fc/Fc ligand complex. Fc ligands include but are notlimited to FcγRs, FcγRs, FcγRs, FcRn, C1q, C3, mannan binding lectin,mannose receptor, staphylococcal protein A, streptococcal protein G, andviral FcγR. Fc ligands also include Fc receptor homologs (FcRH), whichare a family of Fc receptors that are homologous to the FcγRs (Davis etal., 2002, Immunological Reviews 190:123-136, entirely incorporated byreference). Fc ligands may include undiscovered molecules that bind Fc.Particular IgG Fc ligands are FcRn and Fc gamma receptors. By “Fcligand” as used herein is meant a molecule, preferably a polypeptide,from any organism that binds to the Fc region of an antibody to form anFc/Fc ligand complex.

By “Fc gamma receptor”, “FcγR” or “FcgammaR” as used herein is meant anymember of the family of proteins that bind the IgG antibody Fc regionand is encoded by an FcγR gene. In humans this family includes but isnot limited to FcγRI (CD64), including isoforms FcγRIa, FcγRIb, andFcγRIc; FcγRII (CD32), including isoforms FcγRIIa (including allotypesH131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), andFcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa (includingallotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIIb-NA1and FcγRIIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65,entirely incorporated by reference), as well as any undiscovered humanFcγRs or FcγR isoforms or allotypes. An FcγR may be from any organism,including but not limited to humans, mice, rats, rabbits, and monkeys.Mouse FcγRs include but are not limited to FcγRI (CD64), FcγRII (CD32),FcγRIII (CD16), and FcγRIII-2 (CD16-2), as well as any undiscoveredmouse FcγRs or FcγR isoforms or allotypes.

By “FcRn” or “neonatal Fc Receptor” as used herein is meant a proteinthat binds the IgG antibody Fc region and is encoded at least in part byan FcRn gene. The FcRn may be from any organism, including but notlimited to humans, mice, rats, rabbits, and monkeys. As is known in theart, the functional FcRn protein comprises two polypeptides, oftenreferred to as the heavy chain and light chain. The light chain isbeta-2-microglobulin and the heavy chain is encoded by the FcRn gene.Unless other wise noted herein, FcRn or an FcRn protein refers to thecomplex of FcRn heavy chain with beta-2-microglobulin. Sequences ofparticular interest of FcRn are shown in the Figures, particularly thehuman species.

By “parent polypeptide” as used herein is meant an unmodifiedpolypeptide that is subsequently modified to generate a variant. Theparent polypeptide may be a naturally occurring polypeptide, or avariant or engineered version of a naturally occurring polypeptide.Parent polypeptide may refer to the polypeptide itself, compositionsthat comprise the parent polypeptide, or the amino acid sequence thatencodes it. Accordingly, by “parent immunoglobulin” as used herein ismeant an unmodified immunoglobulin polypeptide that is modified togenerate a variant, and by “parent antibody” as used herein is meant anunmodified antibody that is modified to generate a variant antibody. Itshould be noted that “parent antibody” includes known commercial,recombinantly produced antibodies as outlined below.

By “position” as used herein is meant a location in the sequence of aprotein. Positions may be numbered sequentially, or according to anestablished format, for example the EU index for antibody numbering.

By “target antigen” as used herein is meant the molecule that is boundspecifically by the variable region of a given antibody. A targetantigen may be a protein, carbohydrate, lipid, or other chemicalcompound.

By “target cell” as used herein is meant a cell that expresses a targetantigen.

By “variable region” as used herein is meant the region of animmunoglobulin that comprises one or more Ig domains substantiallyencoded by any of the Vκ, Vλ, and/or VH genes that make up the kappa,lambda, and heavy chain immunoglobulin genetic loci respectively.

By “wild type or WT” herein is meant an amino acid sequence or anucleotide sequence that is found in nature, including allelicvariations. A WT protein has an amino acid sequence or a nucleotidesequence that has not been intentionally modified.

The present invention is directed to antibodies that exhibit increasedbinding to FcRn relative to a wild-type antibody. For example, in someinstances, increased binding results in cellular recycling of theantibody and hence increased half-life. In addition, antibodiesexhibiting increased binding to FcRn and altered binding to other Fcreceptors, eg. FcγRs, find use in the present invention.

Antibodies

The present application is directed to antibodies that include aminoacid modifications that modulate binding to FcRn. Of particular interestare antibodies that minimally comprise an Fc region, or functionalvariant thereof, that displays increased binding affinity to FcRn atlowered pH, and do not exhibit substantially altered binding at higherpH.

Traditional antibody structural units typically comprise a tetramer.Each tetramer is typically composed of two identical pairs ofpolypeptide chains, each pair having one “light” (typically having amolecular weight of about 25 kDa) and one “heavy” chain (typicallyhaving a molecular weight of about 50-70 kDa). Human light chains areclassified as kappa and lambda light chains. Heavy chains are classifiedas mu, delta, gamma, alpha, or epsilon, and define the antibody'sisotype as IgM, IgD, IgG, IgA, and IgE, respectively. IgG has severalsubclasses, including, but not limited to IgG1, IgG2, IgG3, and IgG4.IgM has subclasses, including, but not limited to, IgM1 and IgM2. Thus,“isotype” as used herein is meant any of the subclasses ofimmunoglobulins defined by the chemical and antigenic characteristics oftheir constant regions. The known human immunoglobulin isotypes areIgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM1, IgM2, IgD, and IgE.

The amino-terminal portion of each chain includes a variable region ofabout 100 to 110 or more amino acids primarily responsible for antigenrecognition. In the variable region, three loops are gathered for eachof the V domains of the heavy chain and light chain to form anantigen-binding site. Each of the loops is referred to as acomplementarity-determining region (hereinafter referred to as a “CDR”),in which the variation in the amino acid sequence is most significant.

The carboxy-terminal portion of each chain defines a constant regionprimarily responsible for effector function. Kabat et al. collectednumerous primary sequences of the variable regions of heavy chains andlight chains. Based on the degree of conservation of the sequences, theyclassified individual primary sequences into the CDR and the frameworkand made a list thereof (see SEQUENCES OF IMMUNOLOGICAL INTEREST, 5thedition, NIH publication, No. 91-3242, E. A. Kabat et al., entirelyincorporated by reference).

In the IgG subclass of immunoglobulins, there are several immunoglobulindomains in the heavy chain. By “immunoglobulin (Ig) domain” herein ismeant a region of an immunoglobulin having a distinct tertiarystructure. Of interest in the present invention are the heavy chaindomains, including, the constant heavy (CH) domains and the hingedomains. In the context of IgG antibodies, the IgG isotypes each havethree CH regions. Accordingly, “CH” domains in the context of IgG are asfollows: “CH1” refers to positions 118-220 according to the EU index asin Kabat. “CH2” refers to positions 237-340 according to the EU index asin Kabat, and “CH3” refers to positions 341-447 according to the EUindex as in Kabat.

Another type of Ig domain of the heavy chain is the hinge region. By“hinge” or “hinge region” or “antibody hinge region” or “immunoglobulinhinge region” herein is meant the flexible polypeptide comprising theamino acids between the first and second constant domains of anantibody. Structurally, the IgG CH1 domain ends at EU position 220, andthe IgG CH2 domain begins at residue EU position 237. Thus for IgG theantibody hinge is herein defined to include positions 221 (D221 in IgG1)to 236 (G236 in IgG1), wherein the numbering is according to the EUindex as in Kabat. In some embodiments, for example in the context of anFc region, the lower hinge is included, with the “lower hinge” generallyreferring to positions 226 or 230.

Of particular interest in the present invention are the Fc regions. By“Fc” or “Fc region”, as used herein is meant the polypeptide comprisingthe constant region of an antibody excluding the first constant regionimmunoglobulin domain and in some cases, part of the hinge. Thus Fcrefers to the last two constant region immunoglobulin domains of IgA,IgD, and IgG, the last three constant region immunoglobulin domains ofIgE and IgM, and the flexible hinge N-terminal to these domains. For IgAand IgM, Fc may include the J chain. For IgG, as illustrated in FIG. 1,Fc comprises immunoglobulin domains Cgamma2 and Cgamma3 (Cg2 and Cg3)and the lower hinge region between Cgamma1 (Cg1) and Cgamma2 (Cg2).Although the boundaries of the Fc region may vary, the human IgG heavychain Fc region is usually defined to include residues C226 or P230 toits carboxyl-terminus, wherein the numbering is according to the EUindex as in Kabat. Fc may refer to this region in isolation, or thisregion in the context of an Fc polypeptide, as described below. By “Fcpolypeptide” as used herein is meant a polypeptide that comprises all orpart of an Fc region. Fc polypeptides include antibodies, Fc fusions,isolated Fcs, and Fc fragments.

In some embodiments, the antibodies are full length. By “full lengthantibody” herein is meant the structure that constitutes the naturalbiological form of an antibody, including variable and constant regions,including one or more modifications as outlined herein.

Alternatively, the antibodies can be a variety of structures, including,but not limited to, antibody fragments, monoclonal antibodies,bispecific antibodies, minibodies, domain antibodies, syntheticantibodies (sometimes referred to herein as “antibody mimetics”),chimeric antibodies, humanized antibodies, antibody fusions (sometimesreferred to as “antibody conjugates”), and fragments of each,respectively.

Antibody Fragments

In one embodiment, the antibody is an antibody fragment. Of particularinterest are antibodies that comprise Fc regions, Fc fusions, and theconstant region of the heavy chain (CH1-hinge-CH2-CH3), again alsoincluding constant heavy region fusions.

Specific antibody fragments include, but are not limited to, (i) the Fabfragment consisting of VL, VH, CL and CH1 domains, (ii) the Fd fragmentconsisting of the VH and CH1 domains, (iii) the Fv fragment consistingof the VL and VH domains of a single antibody; (iv) the dAb fragment(Ward et al., 1989, Nature 341:544-546, entirely incorporated byreference) which consists of a single variable, (v) isolated CDRregions, (vi) F(ab′)2 fragments, a bivalent fragment comprising twolinked Fab fragments (vii) single chain Fv molecules (scFv), wherein aVH domain and a VL domain are linked by a peptide linker which allowsthe two domains to associate to form an antigen binding site (Bird etal., 1988, Science 242:423-426, Huston et al., 1988, Proc. Natl. Acad.Sci. U.S.A. 85:5879-5883, entirely incorporated by reference), (viii)bispecific single chain Fv (WO 03/11161, hereby incorporated byreference) and (ix) “diabodies” or “triabodies”, multivalent ormultispecific fragments constructed by gene fusion (Tomlinson et. al.,2000, Methods Enzymol. 326:461-479; WO94/13804; Holliger et al., 1993,Proc. Natl. Acad. Sci. U.S.A. 90:6444-6448, all entirely incorporated byreference). The antibody fragments may be modified. For example, themolecules may be stabilized by the incorporation of disulphide bridgeslinking the VH and VL domains (Reiter et al., 1996, Nature Biotech.14:1239-1245, entirely incorporated by reference).

Chimeric and Humanized Antibodies

In some embodiments, the scaffold components can be a mixture fromdifferent species. As such, if the protein is an antibody, such antibodymay be a chimeric antibody and/or a humanized antibody. In general, both“chimeric antibodies” and “humanized antibodies” refer to antibodiesthat combine regions from more than one species. For example, “chimericantibodies” traditionally comprise variable region(s) from a mouse (orrat, in some cases) and the constant region(s) from a human. “Humanizedantibodies” generally refer to non-human antibodies that have had thevariable-domain framework regions swapped for sequences found in humanantibodies. Generally, in a humanized antibody, the entire antibody,except the CDRs, is encoded by a polynucleotide of human origin or isidentical to such an antibody except within its CDRs. The CDRs, some orall of which are encoded by nucleic acids originating in a non-humanorganism, are grafted into the beta-sheet framework of a human antibodyvariable region to create an antibody, the specificity of which isdetermined by the engrafted CDRs. The creation of such antibodies isdescribed in, e.g., WO 92/11018, Jones, 1986, Nature 321:522-525,Verhoeyen et al., 1988, Science 239:1534-1536, all entirely incorporatedby reference. “Backmutation” of selected acceptor framework residues tothe corresponding donor residues is often required to regain affinitythat is lost in the initial grafted construct (U.S. Pat. No. 5,530,101;U.S. Pat. No. 5,585,089; U.S. Pat. No. 5,693,761; U.S. Pat. No.5,693,762; U.S. Pat. No. 6,180,370; U.S. Pat. No. 5,859,205; U.S. Pat.No. 5,821,337; U.S. Pat. No. 6,054,297; U.S. Pat. No. 6,407,213, allentirely incorporated by reference). The humanized antibody optimallyalso will comprise at least a portion of an immunoglobulin constantregion, typically that of a human immunoglobulin, and thus willtypically comprise a human Fc region. Humanized antibodies can also begenerated using mice with a genetically engineered immune system. Roqueet al., 2004, Biotechnol. Prog. 20:639-654, entirely incorporated byreference. A variety of techniques and methods for humanizing andreshaping non-human antibodies are well known in the art (See Tsurushita& Vasquez, 2004, Humanization of Monoclonal Antibodies, MolecularBiology of B Cells, 533-545, Elsevier Science (USA), and referencescited therein, all entirely incorporated by reference). Humanizationmethods include but are not limited to methods described in Jones etal., 1986, Nature 321:522-525; Riechmann et al., 1988; Nature332:323-329; Verhoeyen et al., 1988, Science, 239:1534-1536; Queen etal., 1989, Proc Natl Acad Sci, USA 86:10029-33; He et al., 1998, J.Immunol. 160: 1029-1035; Carter et al., 1992, Proc Natl Acad Sci USA89:4285-9, Presta et al., 1997, Cancer Res. 57(20):4593-9; Gorman etal., 1991, Proc. Natl. Acad. Sci. USA 88:4181-4185; O'Connor et al.,1998, Protein Eng 11:321-8, all entirely incorporated by reference.Humanization or other methods of reducing the immunogenicity of nonhumanantibody variable regions may include resurfacing methods, as describedfor example in Roguska et al., 1994, Proc. Natl. Acad. Sci. USA91:969-973, entirely incorporated by reference. In one embodiment, theparent antibody has been affinity matured, as is known in the art.Structure-based methods may be employed for humanization and affinitymaturation, for example as described in U.S. Ser. No. 11/004,590.Selection based methods may be employed to humanize and/or affinitymature antibody variable regions, including but not limited to methodsdescribed in Wu et al., 1999, J. Mol. Biol. 294:151-162; Baca et al.,1997, J. Biol. Chem. 272(16):10678-10684; Rosok et al., 1996, J. Biol.Chem. 271(37): 22611-22618; Rader et al., 1998, Proc. Natl. Acad. Sci.USA 95: 8910-8915; Krauss et al., 2003, Protein Engineering16(10):753-759, all entirely incorporated by reference. Otherhumanization methods may involve the grafting of only parts of the CDRs,including but not limited to methods described in U.S. Ser. No.09/810,510; Tan et al., 2002, J. Immunol. 169:1119-1125; De Pascalis etal., 2002, J. Immunol. 169:3076-3084, all entirely incorporated byreference.

Bispecific Antibodies

In one embodiment, the antibodies of the invention multispecificantibody, and notably a bispecific antibody, also sometimes referred toas “diabodies”. These are antibodies that bind to two (or more)different antigens. Diabodies can be manufactured in a variety of waysknown in the art (Holliger and Winter, 1993, Current Opinion Biotechnol.4:446-449, entirely incorporated by reference), e.g., preparedchemically or from hybrid hybridomas.

Minibodies

In one embodiment, the antibody is a minibody. Minibodies are minimizedantibody-like proteins comprising a scFv joined to a CH3 domain. Hu etal., 1996, Cancer Res. 56:3055-3061, entirely incorporated by reference.In some cases, the scFv can be joined to the Fc region, and may includesome or the entire hinge region.

Antibody Fusions

In one embodiment, the antibodies of the invention are antibody fusionproteins (sometimes referred to herein as an “antibody conjugate”). Onetype of antibody fusions comprises Fc fusions, which join the Fc regionwith a conjugate partner. By “Fc fusion” as used herein is meant aprotein wherein one or more polypeptides is operably linked to an Fcregion. Fc fusion is herein meant to be synonymous with the terms“immunoadhesin”, “Ig fusion”, “Ig chimera”, and “receptor globulin”(sometimes with dashes) as used in the prior art (Chamow et al., 1996,Trends Biotechnol 14:52-60; Ashkenazi et al., 1997, Curr Opin Immunol9:195-200, both entirely incorporated by reference). An Fc fusioncombines the Fc region of an immunoglobulin with a fusion partner, whichin general can be any protein or small molecule. Virtually any proteinor small molecule may be linked to Fc to generate an Fc fusion. Proteinfusion partners may include, but are not limited to, the variable regionof any antibody, the target-binding region of a receptor, an adhesionmolecule, a ligand, an enzyme, a cytokine, a chemokine, or some otherprotein or protein domain. Small molecule fusion partners may includeany therapeutic agent that directs the Fc fusion to a therapeutictarget. Such targets may be any molecule, preferably an extracellularreceptor, which is implicated in disease. Thus, the IgG variants can belinked to one or more fusion partners. In one alternate embodiment, theIgG variant is conjugated or operably linked to another therapeuticcompound. The therapeutic compound may be a cytotoxic agent, achemotherapeutic agent, a toxin, a radioisotope, a cytokine, or othertherapeutically active agent. The IgG may be linked to one of a varietyof nonproteinaceous polymers, e.g., polyethylene glycol, polypropyleneglycol, polyoxyalkylenes, or copolymers of polyethylene glycol andpolypropylene glycol.

In addition to Fc fusions, antibody fusions include the fusion of theconstant region of the heavy chain with one or more fusion partners(again including the variable region of any antibody), while otherantibody fusions are substantially or completely full length antibodieswith fusion partners. In one embodiment, a role of the fusion partner isto mediate target binding, and thus it is functionally analogous to thevariable regions of an antibody (and in fact can be). Virtually anyprotein or small molecule may be linked to Fc to generate an Fc fusion(or antibody fusion). Protein fusion partners may include, but are notlimited to, the target-binding region of a receptor, an adhesionmolecule, a ligand, an enzyme, a cytokine, a chemokine, or some otherprotein or protein domain. Small molecule fusion partners may includeany therapeutic agent that directs the Fc fusion to a therapeutictarget. Such targets may be any molecule, preferably an extracellularreceptor, which is implicated in disease.

The conjugate partner can be proteinaceous or non-proteinaceous; thelatter generally being generated using functional groups on the antibodyand on the conjugate partner. For example linkers are known in the art;for example, homo- or hetero-bifunctional linkers as are well known(see, 1994 Pierce Chemical Company catalog, technical section oncross-linkers, pages 155-200, incorporated herein by reference).

Suitable conjugates include, but are not limited to, labels as describedbelow, drugs and cytotoxic agents including, but not limited to,cytotoxic drugs (e.g., chemotherapeutic agents) or toxins or activefragments of such toxins. Suitable toxins and their correspondingfragments include diptheria A chain, exotoxin A chain, ricin A chain,abrin A chain, curcin, crotin, phenomycin, enomycin and the like.Cytotoxic agents also include radiochemicals made by conjugatingradioisotopes to antibodies, or binding of a radionuclide to a chelatingagent that has been covalently attached to the antibody. Additionalembodiments utilize calicheamicin, auristatins, geldanamycin,maytansine, and duocarmycins and analogs; for the latter, see U.S.2003/0050331A1, entirely incorporated by reference.

Covalent Modifications of Antibodies

Covalent modifications of antibodies are included within the scope ofthis invention, and are generally, but not always, donepost-translationally. For example, several types of covalentmodifications of the antibody are introduced into the molecule byreacting specific amino acid residues of the antibody with an organicderivatizing agent that is capable of reacting with selected side chainsor the N- or C-terminal residues.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesmay also be derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole and the like.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing alpha-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pKa of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidazole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using 125I or 131I to prepare labeled proteinsfor use in radioimmunoassay, the chloramine T method described abovebeing suitable.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R′—N═C═N—R′), where R and R′ are optionallydifferent alkyl groups, such as1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore,aspartyl and glutamyl residues are converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Derivatization with bifunctional agents is useful for crosslinkingantibodies to a water-insoluble support matrix or surface for use in avariety of methods, in addition to methods described below. Commonlyused crosslinking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis (succinimidylpropionate), and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane. Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440, all entirely incorporated by reference, are employed forprotein immobilization.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively.Alternatively, these residues are deamidated under mildly acidicconditions. Either form of these residues falls within the scope of thisinvention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W. H. Freeman & Co., San Francisco, pp. 79-86 [1983],entirely incorporated by reference), acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

Glycosylation

Another type of covalent modification is glycosylation. In anotherembodiment, the IgG variants disclosed herein can be modified to includeone or more engineered glycoforms. By “engineered glycoform” as usedherein is meant a carbohydrate composition that is covalently attachedto an IgG, wherein said carbohydrate composition differs chemically fromthat of a parent IgG. Engineered glycoforms may be useful for a varietyof purposes, including but not limited to enhancing or reducing effectorfunction. Engineered glycoforms may be generated by a variety of methodsknown in the art (Umaña et al., 1999, Nat Biotechnol 17:176-180; Davieset al., 2001, Biotechnol Bioeng 74:288-294; Shields et al., 2002, J BiolChem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473;U.S. Pat. No. 6,602,684; U.S. Ser. No. 10/277,370; U.S. Ser. No.10/113,929; PCT WO 00/61739A1; PCT WO 01/29246A1; PCT WO 02/31140A1; PCTWO 02/30954A1, all entirely incorporated by reference; (Potelligent®technology [Biowa, Inc., Princeton, N.J.]; GlycoMAb® glycosylationengineering technology [Glycart Biotechnology AG, Zürich, Switzerland]).Many of these techniques are based on controlling the level offucosylated and/or bisecting oligosaccharides that are covalentlyattached to the Fc region, for example by expressing an IgG in variousorganisms or cell lines, engineered or otherwise (for example Lec-13 CHOcells or rat hybridoma YB2/0 cells), by regulating enzymes involved inthe glycosylation pathway (for example FUT8 [α1,6-fucosyltranserase]and/or β1-4-N-acetylglucosaminyltransferase III [GnTIII]), or bymodifying carbohydrate(s) after the IgG has been expressed. Engineeredglycoform typically refers to the different carbohydrate oroligosaccharide; thus an IgG variant, for example an antibody or Fcfusion, can include an engineered glycoform. Alternatively, engineeredglycoform may refer to the IgG variant that comprises the differentcarbohydrate or oligosaccharide. As is known in the art, glycosylationpatterns can depend on both the sequence of the protein (e.g., thepresence or absence of particular glycosylation amino acid residues,discussed below), or the host cell or organism in which the protein isproduced. Particular expression systems are discussed below.

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tri-peptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tri-peptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid,most commonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tri-peptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thestarting sequence (for O-linked glycosylation sites). For ease, theantibody amino acid sequence is preferably altered through changes atthe DNA level, particularly by mutating the DNA encoding the targetpolypeptide at preselected bases such that codons are generated thatwill translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on theantibody is by chemical or enzymatic coupling of glycosides to theprotein. These procedures are advantageous in that they do not requireproduction of the protein in a host cell that has glycosylationcapabilities for N- and O-linked glycosylation. Depending on thecoupling mode used, the sugar(s) may be attached to (a) arginine andhistidine, (b) free carboxyl groups, (c) free sulfhydryl groups such asthose of cysteine, (d) free hydroxyl groups such as those of serine,threonine, or hydroxyproline, (e) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan, or (f) the amide group ofglutamine. These methods are described in WO 87/05330 and in Aplin andWriston, 1981, CRC Crit. Rev. Biochem., pp. 259-306, both entirelyincorporated by reference.

Removal of carbohydrate moieties present on the starting antibody may beaccomplished chemically or enzymatically. Chemical deglycosylationrequires exposure of the protein to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving thepolypeptide intact. Chemical deglycosylation is described by Hakimuddinet al., 1987, Arch. Biochem. Biophys. 259:52 and by Edge et al., 1981,Anal. Biochem. 118:131, both entirely incorporated by reference.Enzymatic cleavage of carbohydrate moieties on polypeptides can beachieved by the use of a variety of endo- and exo-glycosidases asdescribed by Thotakura et al., 1987, Meth. Enzymol. 138:350, entirelyincorporated by reference. Glycosylation at potential glycosylationsites may be prevented by the use of the compound tunicamycin asdescribed by Duskin et al., 1982, J. Biol. Chem. 257:3105, entirelyincorporated by reference. Tunicamycin blocks the formation ofprotein-N-glycoside linkages.

Another type of covalent modification of the antibody comprises linkingthe antibody to various nonproteinaceous polymers, including, but notlimited to, various polyols such as polyethylene glycol, polypropyleneglycol or polyoxyalkylenes, in the manner set forth in, for example,2005-2006 PEG Catalog from Nektar Therapeutics (available at the Nektarwebsite) U.S. Pat. No. 4,640,835; 4,496,689; 4,301,144; 4,670,417;4,791,192 or 4,179,337, all entirely incorporated by reference. Inaddition, as is known in the art, amino acid substitutions may be madein various positions within the antibody to facilitate the addition ofpolymers such as PEG. See for example, U.S. Publication No.2005/0114037A1, entirely incorporated by reference.

Labeled Antibodies

In some embodiments, the covalent modification of the antibodies of theinvention comprises the addition of one or more labels. In some cases,these are considered antibody fusions. The term “labelling group” meansany detectable label. In some embodiments, the labelling group iscoupled to the antibody via spacer arms of various lengths to reducepotential steric hindrance. Various methods for labelling proteins areknown in the art and may be used in performing the present invention.

In general, labels fall into a variety of classes, depending on theassay in which they are to be detected: a) isotopic labels, which may beradioactive or heavy isotopes; b) magnetic labels (e.g., magneticparticles); c) redox active moieties; d) optical dyes; enzymatic groups(e.g. horseradish peroxidase, β-galactosidase, luciferase, alkalinephosphatase); e) biotinylated groups; and f) predetermined polypeptideepitopes recognized by a secondary reporter (e.g., leucine zipper pairsequences, binding sites for secondary antibodies, metal bindingdomains, epitope tags, etc.). In some embodiments, the labelling groupis coupled to the antibody via spacer arms of various lengths to reducepotential steric hindrance. Various methods for labelling proteins areknown in the art and may be used in performing the present invention.

Specific labels include optical dyes, including, but not limited to,chromophores, phosphors and fluorophores, with the latter being specificin many instances. Fluorophores can be either “small molecule” fluores,or proteinaceous fluores.

By “fluorescent label” is meant any molecule that may be detected viaits inherent fluorescent properties. Suitable fluorescent labelsinclude, but are not limited to, fluorescein, rhodamine,tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins,pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade BlueJ, TexasRed, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705,Oregon green, the Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430,Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594,Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680), Cascade Blue,Cascade Yellow and R-phycoerythrin (PE) (Molecular Probes, Eugene,Oreg.), FITC, Rhodamine, and Texas Red (Pierce, Rockford, Ill.), Cy5,Cy5.5, Cy7 (Amersham Life Science, Pittsburgh, Pa.). Suitable opticaldyes, including fluorophores, are described in Molecular Probes Handbookby Richard P. Haugland, entirely incorporated by reference.

Suitable proteinaceous fluorescent labels also include, but are notlimited to, green fluorescent protein, including a Renilla, Ptilosarcus,or Aequorea species of GFP (Chalfie et al., 1994, Science 263:802-805),EGFP (Clontech Laboratories, Inc., Genbank Accession Number U55762),blue fluorescent protein (BFP, Quantum Biotechnologies, Inc. 1801 deMaisonneuve Blvd. West, 8th Floor, Montreal, Quebec, Canada H3H 1J9;Stauber, 1998, Biotechniques 24:462-471; Heim et al., 1996, Curr. Biol.6:178-182), enhanced yellow fluorescent protein (EYFP, ClontechLaboratories, Inc.), luciferase (Ichiki et al., 1993, J. Immunol.150:5408-5417), β galactosidase (Nolan et al., 1988, Proc. Natl. Acad.Sci. U.S.A. 85:2603-2607) and Renilla (WO92/15673, WO95/07463,WO98/14605, WO98/26277, WO99/49019, U.S. Pat. Nos. 5,292,658, 5,418,155,5,683,888, 5,741,668, 5,777,079, 5,804,387, 5874304, 5876995, 5925558).All of the above-cited references in this paragraph are expresslyincorporated herein by reference.

IgG Variants

In one embodiment, the invention provides variant IgG proteins. At aminimum, IgG variants comprise an antibody fragment comprising theCH2-CH3 region of the heavy chain. In addition, suitable IgG variantscomprise Fc domains (e.g. including the lower hinge region), as well asIgG variants comprising the constant region of the heavy chain(CH1-hinge-CH2-CH3) also being useful in the present invention, all ofwhich can be fused to fusion partners.

An IgG variant includes one or more amino acid modifications relative toa parent IgG polypeptide, in some cases relative to the wild type IgG.The IgG variant can have one or more optimized properties. An IgGvariant differs in amino acid sequence from its parent IgG by virtue ofat least one amino acid modification. Thus IgG variants have at leastone amino acid modification compared to the parent. Alternatively, theIgG variants may have more than one amino acid modification as comparedto the parent, for example from about one to fifty amino acidmodifications, preferably from about one to ten amino acidmodifications, and most preferably from about one to about five aminoacid modifications compared to the parent.

Thus the sequences of the IgG variants and those of the parent Fcpolypeptide are substantially homologous. For example, the variant IgGvariant sequences herein will possess about 80% homology with the parentIgG variant sequence, preferably at least about 90% homology, and mostpreferably at least about 95% homology. Modifications may be madegenetically using molecular biology, or may be made enzymatically orchemically.

Target Antigens for Antibodies

Virtually any antigen may be targeted by the IgG variants, including butnot limited to proteins, subunits, domains, motifs, and/or epitopesbelonging to the following list of target antigens, which includes bothsoluble factors such as cytokines and membrane-bound factors, includingtransmembrane receptors: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a,8-oxo-dG, A1 Adenosine Receptor, A33, ACE, ACE-2, Activin, Activin A,Activin AB, Activin B, Activin C, Activin RIA, Activin RIA ALK-2,Activin RIB ALK-4, Activin RIIA, Activin RIIB, ADAM, ADAM10, ADAM12,ADAM15, ADAM17/TACE, ADAM8, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, Addressins,aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1-antitrypsin, alpha-V/beta-1antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART,Artemin, anti-Id, ASPARTIC, Atrial natriuretic factor, av/b3 integrin,Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte Stimulator (BlyS), BACE,BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA,BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a,BMP-3 Osteogenin, BMP-4 BMP-2b, BMP-5, BMP-6 Vgr-1, BMP-7 (OP-1), BMP-8(BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1,BMPR-II (BRK-3), BMPs, b-NGF, BOK, Bombesin, Bone-derived neurotrophicfactor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a,C10, CA125, CAD-8, Calcitonin, cAMP, carcinoembryonic antigen (CEA),carcinoma-associated antigen, Cathepsin A, Cathepsin B, CathepsinC/DPPI, Cathepsin D, Cathepsin E, Cathepsin H, Cathepsin L, Cathepsin O,Cathepsin S, Cathepsin V, Cathepsin X/Z/P, CBL, CCI, CCK2, CCL, CCL1,CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2,CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3,CCL4, CCL5, CCL6, CCL7, CCL8, CCL9/10, CCR, CCR1, CCR10, CCR10, CCR2,CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5,CD6, CD7, CD8, CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18,CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L,CD32, CD33 (p67 proteins), CD34, CD38, CD40, CD40L, CD44, CD45, CD46,CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1),CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152,CD164, CEACAM5, CFTR, cGMP, CINC, Clostridium botulinum toxin,Clostridium perfringens toxin, CKb8-1, CLC, CMV, CMV UL, CNTF, CNTN-1,COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL,CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCL10,CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2,CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-associated antigen, DAN,DCC, DcR3, DC-SIGN, Decay accelerating factor, des(1-3)-IGF-I (brainIGF-1), Dhh, digoxin, DNAM-1, Dnase, Dpp, DPPIV/CD26, Dtk, ECAD, EDA,EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelinreceptor, Enkephalinase, eNOS, Eot, eotaxin1, EpCAM, Ephrin B2/EphB4,EPO, ERCC, E-selectin, ET-1, Factor IIa, Factor VII, Factor VIIIc,Factor IX, fibroblast activation protein (FAP), Fas, FcR1, FEN-1,Ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, Fibrin, FL,FLIP, Flt-3, Flt-4, Follicle stimulating hormone, Fractalkine, FZD1,FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas 6,GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14,CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8(Myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1,GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, Glucagon, Glut 4, glycoproteinIIb/IIIa (GP IIb/IIIa), GM-CSF, gp130, gp72, GRO, Growth hormonereleasing factor, Hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMV gBenvelope glycoprotein, HCMV) gH envelope glycoprotein, HCMV UL,Hemopoietic growth factor (HGF), Hep B gp120, heparanase, Her2, Her2/neu(ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gBglycoprotein, HSV gD glycoprotein, HGFA, High molecular weightmelanoma-associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp120 V3loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin,human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, I-309,IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF,IGF binding proteins, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R,IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10,IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon (INF)-alpha,INF-beta, INF-gamma, Inhibin, iNOS, Insulin A-chain, Insulin B-chain,Insulin-like growth factor 1, integrin alpha2, integrin alpha3, integrinalpha4, integrin alpha4/beta1, integrin alpha4/beta7, integrin alpha5(alphaV), integrin alpha5/beta1, integrin alpha5/beta3, integrin alpha6,integrin beta1, integrin beta2, interferon gamma, IP-10, I-TAC, JE,Kallikrein 2, Kallikrein 5, Kallikrein 6, Kallikrein 11, Kallikrein 12,Kallikrein 14, Kallikrein 15, Kallikrein L1, Kallikrein L2, KallikreinL3, Kallikrein L4, KC, KDR, Keratinocyte Growth Factor (KGF), laminin 5,LAMP, LAP, LAP (TGF-1), Latent TGF-1, Latent TGF-1 bpi, LBP, LDGF,LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3,Lfo, LIF, LIGHT, lipoproteins, LIX, LKN, Lptn, L-Selectin, LT-a, LT-b,LTB4, LTBP-1, Lung surfactant, Luteinizing hormone, Lymphotoxin BetaReceptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF,MDC, Mer, METALLOPROTEASES, MGDF receptor, MGMT, MHC(HLA-DR), MIF, MIG,MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13,MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo,MSK, MSP, mucin (Mud), MUC18, Muellerian-inhibitin substance, Mug, MuSK,NAIP, NAP, NCAD, N-Cadherin, NCA 90, NCAM, NCAM, Neprilysin,Neurotrophin-3, -4, or -6, Neurturin, Neuronal growth factor (NGF),NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN,OSM, OX40L, OX40R, p150, p95, PADPr, Parathyroid hormone, PARC, PARP,PBR, PBSF, PCAD, P-Cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4,PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP),P1GF, PLP, PP14, Proinsulin, Prorelaxin, Protein C, PS, PSA, PSCA,prostate specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51,RANK, RANKL, RANTES, RANTES, Relaxin A-chain, Relaxin B-chain, renin,respiratory syncytial virus (RSV) F, RSV Fgp, Ret, Rheumatoid factors,RLIP76, RPA2, RSK, S100, SCF/KL, SDF-1, SERINE, Serum albumin, sFRP-3,Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat,STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72),TARC, TCA-3, T-cell receptors (e.g., T-cell receptor alpha/beta), TdT,TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkalinephosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan Specific,TGF-beta R1 (ALK-5), TGF-beta RII, TGF-beta RIIb, TGF-beta RIII,TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5, Thrombin, ThymusCk-1, Thyroid stimulating hormone, Tie, TIMP, TIQ, Tissue Factor,TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha beta, TNF-beta2, TNFc,TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1Apo-2, DR4), TNFRSF10B (TRAIL R2DR5,KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3DcR1, LIT, TRID),TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R),TNFRSF11B (OPG OCIF, TR1), TNFRSF12 (TWEAK R FN14), TNFRSF13B (TACI),TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF16(NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR AITR), TNFRSF19 (TROYTAJ, TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF RI CD120a, p55-60),TNFRSF1B (TNF RII CD120b, p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNFRIII, TNFC R), TNFRSF4 (OX40 ACT35, TXGP1 R), TNFRSF5 (CD40 p50),TNFRSF6 (Fas Apo-1, APT1, CD95), TNFRSF6B (DcR3M68, TR6), TNFRSF7(CD27), TNFRSF8 (CD30), TNFRSF9 (4-1BB CD137, ILA), TNFRSF21 (DR6),TNFRSF22 (DcTRAIL R2TNFRH2), TNFRST23 (DcTRAIL R1 TNFRH1), TNFRSF25 (DR3Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 Ligand, TL2),TNFSF11 (TRANCE/RANK Ligand ODF, OPG Ligand), TNFSF12 (TWEAK Apo-3Ligand, DR3 Ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1,THANK, TNFSF20), TNFSF14 (LIGHT HVEM Ligand, LTg), TNFSF15 (TL1A/VEGI),TNFSF18 (GITR Ligand AITR Ligand, TL6), TNFSF1A (TNF-a Conectin, DIF,TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4(OX40 Ligand gp34, TXGP1), TNFSF5 (CD40 Ligand CD154, gp39, HIGM1, IMD3,TRAP), TNFSF6 (Fas Ligand Apo-1 Ligand, APT1 Ligand), TNFSF7 (CD27Ligand CD70), TNFSF8 (CD30 Ligand CD153), TNFSF9 (4-1BB Ligand CD137Ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE,transferring receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associatedantigen CA 125, tumor-associated antigen expressing Lewis Y relatedcarbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, Urokinase, VCAM, VCAM-1,VECAD, VE-Cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3(flt-4), VEGI, VIM, Viral antigens, VLA, VLA-1, VLA-4, VNR integrin, vonWillebrands factor, WI F-1, WNT1, WNT2, WNT2B/13, WNT3, WNT3A, WNT4,WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B,WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD,and receptors for hormones and growth factors.

One skilled in the art will appreciate that the aforementioned list oftargets refers not only to specific proteins and biomolecules, but thebiochemical pathway or pathways that comprise them. For example,reference to CTLA-4 as a target antigen implies that the ligands andreceptors that make up the T cell co-stimulatory pathway, includingCTLA-4, B7-1, B7-2, CD28, and any other undiscovered ligands orreceptors that bind these proteins, are also targets. Thus target asused herein refers not only to a specific biomolecule, but the set ofproteins that interact with said target and the members of thebiochemical pathway to which said target belongs. One skilled in the artwill further appreciate that any of the aforementioned target antigens,the ligands or receptors that bind them, or other members of theircorresponding biochemical pathway, may be operably linked to the Fcvariants of the present invention in order to generate an Fc fusion.Thus for example, an Fc fusion that targets EGFR could be constructed byoperably linking an Fc variant to EGF, TGF-b, or any other ligand,discovered or undiscovered, that binds EGFR. Accordingly, an Fc variantof the present invention could be operably linked to EGFR in order togenerate an Fc fusion that binds EGF, TGF-b, or any other ligand,discovered or undiscovered, that binds EGFR. Thus virtually anypolypeptide, whether a ligand, receptor, or some other protein orprotein domain, including but not limited to the aforementioned targetsand the proteins that compose their corresponding biochemical pathways,may be operably linked to the Fc variants of the present invention todevelop an Fc fusion.

The choice of suitable antigen depends on the desired application. Foranti-cancer treatment it is desirable to have a target whose expressionis restricted to the cancerous cells. Some targets that have provenespecially amenable to antibody therapy are those with signalingfunctions. Other therapeutic antibodies exert their effects by blockingsignaling of the receptor by inhibiting the binding between a receptorand its cognate ligand. Another mechanism of action of therapeuticantibodies is to cause receptor down regulation. Other antibodies do notwork by signaling through their target antigen. In some cases,antibodies directed against infectious disease agents are used.

In one embodiment, the Fc variants of the present invention areincorporated into an antibody against a cytokine. Alternatively, the Fcvariants are fused or conjugated to a cytokine. By “cytokine” as usedherein is meant a generic term for proteins released by one cellpopulation that act on another cell as intercellular mediators. Forexample, as described in Penichet et al., 2001, J Immunol Methods248:91-101, expressly incorporated by reference, cytokines may be fusedto antibody to provide an array of desirable properties. Examples ofsuch cytokines are lymphokines, monokines, and traditional polypeptidehormones. Included among the cytokines are growth hormone such as humangrowth hormone, N-methionyl human growth hormone, and bovine growthhormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin;prorelaxin; glycoprotein hormones such as follicle stimulating hormone(FSH), thyroid stimulating hormone (TSH), and luteinizing hormone (LH);hepatic growth factor; fibroblast growth factor; prolactin; placentallactogen; tumor necrosis factor-alpha and -beta; mullerian-inhibitingsubstance; mouse gonadotropin-associated peptide; inhibin; activin;vascular endothelial growth factor; integrin; thrombopoietin (TPO);nerve growth factors such as NGF-beta; platelet-growth factor;transforming growth factors (TGFs) such as TGF-alpha and TGF-beta;insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-alpha, beta, and-gamma; colony stimulating factors (CSFs) such as macrophage-CSF(M-CSF); granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF(G-CSF); interleukins (ILs) such as IL-1, IL-1alpha, IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; IL-15, a tumornecrosis factor such as TNF-alpha or TNF-beta; C5a; and otherpolypeptide factors including LIF and kit ligand (KL). As used herein,the term cytokine includes proteins from natural sources or fromrecombinant cell culture, and biologically active equivalents of thenative sequence cytokines.

Cytokines and soluble targets, such as TNF superfamily members, arepreferred targets for use with the variants of the present invention.For example, anti-VEGF, anti-CTLA-4, and anti-TNF antibodies, orfragments thereof, are particularly good antibodies for the use of Fcvariants that increase the FcRn binding. Therapeutics against thesetargets are frequently involved in the treatment of autoimmune diseasesand require multiple injections over long time periods. Therefore,longer serum half-lives and less frequent treatments, brought about fromthe variants of the present invention, are particularly preferred.

A number of antibodies and Fc fusions that are approved for use, inclinical trials, or in development may benefit from the Fc variants ofthe present invention. These antibodies and Fc fusions are hereinreferred to as “clinical products and candidates”. Thus in a preferredembodiment, the Fc polypeptides of the present invention may find use ina range of clinical products and candidates. For example, a number ofantibodies that target CD20 may benefit from the Fc polypeptides of thepresent invention. For example the Fc polypeptides of the presentinvention may find use in an antibody that is substantially similar torituximab (Rituxan®, IDEC/Genentech/Roche) (see for example U.S. Pat.No. 5,736,137), a chimeric anti-CD20 antibody approved to treatNon-Hodgkin's lymphoma; HuMax-CD20, an anti-CD20 currently beingdeveloped by Genmab, an anti-CD20 antibody described in U.S. Pat. No.5,500,362, AME-133 (Applied Molecular Evolution), hA20 (Immunomedics,Inc.), HumaLYM (Intracel), and PRO70769 (PCT/US2003/040426, entitled“Immunoglobulin Variants and Uses Thereof”). A number of antibodies thattarget members of the family of epidermal growth factor receptors,including EGFR (ErbB-1), Her2/neu (ErbB-2), Her3 (ErbB-3), Her4(ErbB-4), may benefit from the Fc polypeptides of the present invention.For example the Fc polypeptides of the present invention may find use inan antibody that is substantially similar to trastuzumab (Herceptin®,Genentech) (see for example U.S. Pat. No. 5,677,171), a humanizedanti-Her2/neu antibody approved to treat breast cancer; pertuzumab(rhuMab-2C4, Omnitarg™), currently being developed by Genentech; ananti-Her2 antibody described in U.S. Pat. No. 4,753,894; cetuximab(Erbitux®, Imclone) (U.S. Pat. No. 4,943,533; PCT WO 96/40210), achimeric anti-EGFR antibody in clinical trials for a variety of cancers;ABX-EGF (U.S. Pat. No. 6,235,883), currently being developed byAbgenix-Immunex-Amgen; HuMax-EGFr (U.S. Ser. No. 10/172,317), currentlybeing developed by Genmab; 425, EMD55900, EMD62000, and EMD72000 (MerckKGaA) (U.S. Pat. No. 5,558,864; Murthy et al. 1987, Arch BiochemBiophys. 252(2):549-60; Rodeck et al., 1987, J Cell Biochem.35(4):315-20; Kettleborough et al., 1991, Protein Eng. 4(7):773-83);ICR62 (Institute of Cancer Research) (PCT WO 95/20045; Modjtahedi etal., 1993, J. Cell Biophys. 1993, 22(1-3):129-46; Modjtahedi et al.,1993, Br J. Cancer. 1993, 67(2):247-53; Modjtahedi et al, 1996, Br JCancer, 73(2):228-35; Modjtahedi et al, 2003, Int J Cancer,105(2):273-80); TheraCIM hR3 (YM Biosciences, Canada and Centro deImmunologia Molecular, Cuba (U.S. Pat. No. 5,891,996; U.S. Pat. No.6,506,883; Mateo et al, 1997, Immunotechnology, 3(1):71-81); mAb-806(Ludwig Institue for Cancer Research, Memorial Sloan-Kettering)(Jungbluth et al. 2003, Proc Natl Acad Sci USA. 100(2):639-44); KSB-102(KS Biomedix); MR1-1 (IVAX, National Cancer Institute) (PCT WO0162931A2); and SC100 (Scancell) (PCT WO 01/88138). In another preferredembodiment, the Fc polypeptides of the present invention may find use inalemtuzumab (Campath®, Millenium), a humanized monoclonal antibodycurrently approved for treatment of B-cell chronic lymphocytic leukemia.The Fc polypeptides of the present invention may find use in a varietyof antibodies or Fc fusions that are substantially similar to otherclinical products and candidates, including but not limited tomuromonab-CD3 (Orthoclone OKT3®), an anti-CD3 antibody developed byOrtho Biotech/Johnson & Johnson, ibritumomab tiuxetan (Zevalin®), ananti-CD20 antibody developed by IDEC/Schering AG, gemtuzumab ozogamicin(Mylotarg®), an anti-CD33 (p67 protein) antibody developed byCelltech/Wyeth, alefacept (Amevive®), an anti-LFA-3 Fc fusion developedby Biogen), abciximab (ReoPro®), developed by Centocor/Lilly,basiliximab (Simulect®), developed by Novartis, palivizumab (Synagis®),developed by MedImmune, infliximab (Remicade®), an anti-TNFalphaantibody developed by Centocor, adalimumab (Humira®), an anti-TNFalphaantibody developed by Abbott, Humicade™ an anti-TNFalpha antibodydeveloped by Celltech, etanercept (Enbrel®), an anti-TNFalpha Fc fusiondeveloped by Immunex/Amgen, ABX-CBL, an anti-CD147 antibody beingdeveloped by Abgenix, ABX-IL8, an anti-IL8 antibody being developed byAbgenix, ABX-MA1, an anti-MUC18 antibody being developed by Abgenix,Pemtumomab (R1549, 90Y-muHMFG1), an anti-MUC1 In development byAntisoma, Therex (R1550), an anti-MUC1 antibody being developed byAntisoma, AngioMab (AS1405), being developed by Antisoma, HuBC-1, beingdeveloped by Antisoma, Thioplatin (AS1407) being developed by Antisoma,Antegren® (natalizumab), an anti-alpha-4-beta-1 (VLA-4) andalpha-4-beta-7 antibody being developed by Biogen, VLA-1 mAb, ananti-VLA-1 integrin antibody being developed by Biogen, LTBR mAb, ananti-lymphotoxin beta receptor (LTBR) antibody being developed byBiogen, CAT-152, an anti-TGF-β2 antibody being developed by CambridgeAntibody Technology, J695, an anti-IL-12 antibody being developed byCambridge Antibody Technology and Abbott, CAT-192, an anti-TGFβ1antibody being developed by Cambridge Antibody Technology and Genzyme,CAT-213, an anti-Eotaxin1 antibody being developed by Cambridge AntibodyTechnology, LymphoStat-B™ an anti-Blys antibody being developed byCambridge Antibody Technology and Human Genome Sciences Inc., TRAIL-R1mAb, an anti-TRAIL-R1 antibody being developed by Cambridge AntibodyTechnology and Human Genome Sciences, Inc., Avastin™ (bevacizumab,rhuMAb-VEGF), an anti-VEGF antibody being developed by Genentech, ananti-HER receptor family antibody being developed by Genentech,Anti-Tissue Factor (ATF), an anti-Tissue Factor antibody being developedby Genentech, Xolair™ (Omalizumab), an anti-IgE antibody being developedby Genentech, Raptiva™ (Efalizumab), an anti-CD11a antibody beingdeveloped by Genentech and Xoma, MLN-02 Antibody (formerly LDP-02),being developed by Genentech and Millenium Pharmaceuticals, HuMax CD4,an anti-CD4 antibody being developed by Genmab, HuMax-IL15, an anti-IL15antibody being developed by Genmab and Amgen, HuMax-Inflam, beingdeveloped by Genmab and Medarex, HuMax-Cancer, an anti-Heparanase Iantibody being developed by Genmab and Medarex and Oxford GcoSciences,HuMax-Lymphoma, being developed by Genmab and Amgen, HuMax-TAC, beingdeveloped by Genmab, IDEC-131, and anti-CD40L antibody being developedby IDEC Pharmaceuticals, IDEC-151 (Clenoliximab), an anti-CD4 antibodybeing developed by IDEC Pharmaceuticals, IDEC-114, an anti-CD80 antibodybeing developed by IDEC Pharmaceuticals, IDEC-152, an anti-CD23 beingdeveloped by IDEC Pharmaceuticals, anti-macrophage migration factor(MIF) antibodies being developed by IDEC Pharmaceuticals, BEC2, ananti-idiotypic antibody being developed by Imclone, IMC-1C11, ananti-KDR antibody being developed by Imclone, DC101, an anti-flk-1antibody being developed by Imclone, anti-VE cadherin antibodies beingdeveloped by Imclone, CEA-Cide™ (labetuzumab), an anti-carcinoembryonicantigen (CEA) antibody being developed by Immunomedics, LymphoCide™(Epratuzumab), an anti-CD22 antibody being developed by Immunomedics,AFP-Cide, being developed by Immunomedics, MyelomaCide, being developedby Immunomedics, LkoCide, being developed by Immunomedics, ProstaCide,being developed by Immunomedics, MDX-010, an anti-CTLA4 antibody beingdeveloped by Medarex, MDX-060, an anti-CD30 antibody being developed byMedarex, MDX-070 being developed by Medarex, MDX-018 being developed byMedarex, Osidem™ (IDM-1), and anti-Her2 antibody being developed byMedarex and Immuno-Designed Molecules, HuMax™-CD4, an anti-CD4 antibodybeing developed by Medarex and Genmab, HuMax-IL15, an anti-IL15 antibodybeing developed by Medarex and Genmab, CNTO 148, an anti-TNFα antibodybeing developed by Medarex and Centocor/J&J, CNTO 1275, an anti-cytokineantibody being developed by Centocor/J&J, MOR101 and MOR102,anti-intercellular adhesion molecule-1 (ICAM-1) (CD54) antibodies beingdeveloped by MorphoSys, MOR201, an anti-fibroblast growth factorreceptor 3 (FGFR-3) antibody being developed by MorphoSys, Nuvion®(visilizumab), an anti-CD3 antibody being developed by Protein DesignLabs, HuZAF™, an anti-gamma interferon antibody being developed byProtein Design Labs, Anti-α5β1 Integrin, being developed by ProteinDesign Labs, anti-IL-12, being developed by Protein Design Labs, ING-1,an anti-Ep-CAM antibody being developed by Xoma, and MLN01, ananti-Beta2 integrin antibody being developed by Xoma, all of theabove-cited references in this paragraph are expressly incorporatedherein by reference.

The Fc polypeptides of the present invention may be incorporated intothe aforementioned clinical candidates and products, or into antibodiesand Fc fusions that are substantially similar to them. The Fcpolypeptides of the present invention may be incorporated into versionsof the aforementioned clinical candidates and products that arehumanized, affinity matured, engineered, or modified in some other way.

In one embodiment, the Fc polypeptides of the present invention are usedfor the treatment of autoimmune, inflammatory, or transplantindications. Target antigens and clinical products and candidates thatare relevant for such diseases include but are not limited to anti-α4β7integrin antibodies such as LDP-02, anti-beta2 integrin antibodies suchas LDP-01, anti-complement (C5) antibodies such as 5G1.1, anti-CD2antibodies such as BTI-322, MEDI-507, anti-CD3 antibodies such as OKT3,SMART anti-CD3, anti-CD4 antibodies such as IDEC-151, MDX-CD4, OKT4A,anti-CD11a antibodies, anti-CD14 antibodies such as IC14, anti-CD18antibodies, anti-CD23 antibodies such as IDEC 152, anti-CD25 antibodiessuch as Zenapax, anti-CD40L antibodies such as 5c8, Antova, IDEC-131,anti-CD64 antibodies such as MDX-33, anti-CD80 antibodies such asIDEC-114, anti-CD147 antibodies such as ABX-CBL, anti-E-selectinantibodies such as CDP850, anti-gpIIb/IIIa antibodies such asReoPro/Abcixima, anti-ICAM-3 antibodies such as ICM3, anti-ICEantibodies such as VX-740, anti-FcR1 antibodies such as MDX-33, anti-IgEantibodies such as rhuMab-E25, anti-IL-4 antibodies such as SB-240683,anti-IL-5 antibodies such as SB-240563, SCH55700, anti-IL-8 antibodiessuch as ABX-IL8, anti-interferon gamma antibodies, anti-TNF (TNF, TNFa,TNFa, TNF-alpha) antibodies such as CDP571, CDP870, D2E7, Infliximab,MAK-195F, and anti-VLA-4 antibodies such as Antegren.

Fc variants of the present invention such as those with increasedbinding to FcRn may be utilized in TNF inhibitor molecules to provideenhanced properties. Useful TNF inhibitor molecules include any moleculethat inhibits the action of TNF-alpha in a mammal. Suitable examplesinclude the Fc fusion Enbrel® (etanercept) and the antibodies Humira®(adalimumab) and Remicade® (infliximab). Monoclonal antibodies (such asRemicade and Humira) engineered using the Fc variants of the presentinvention to increase FcFn binding, may translate to better efficacythrough an increased half-life.

In some embodiments, antibodies against infectious diseases are used.Antibodies against eukaryotic cells include antibodies targeting yeastcells, including but not limited to Saccharomyces cerevisiae, Hansenulapolymorpha, Kluyveromyces fragilis and K. lactis, Pichia guillerimondiiand P. pastoris, Schizosaccharomyces pombe, plasmodium falciparium, andYarrowia lipolytica.

Antibodies against additional fungal cells are also useful, includingtarget antigens associated with Candida strains including Candidaglabrata, Candida albicans, C. krusei, C. lusitaniae and C. maltosa, aswell as species of Aspergillus, Cryptococcus, Histoplasma, Coccidioides,Blastomyces, and Penicillium, among others

Antibodies directed against target antigens associated with protozoainclude, but are not limited to, antibodies associated with Trypanosoma,Leishmania species including Leishmania donovanii; Plasmodium spp.,Pneumocystis carinii, Cryptosporidium parvum, Giardia lamblia, Entamoebahistolytica, and Cyclospora cayetanensis.

Antibodies against prokaryotic antigens are also useful, includingantibodies against suitable bacteria such as pathogenic andnon-pathogenic prokaryotes including but not limited to Bacillus,including Bacillus anthracis; Vibrio, e.g. V. cholerae; Escherichia,e.g. Enterotoxigenic E. coli, Shigella, e.g. S. dysenteriae; Salmonella,e.g. S. typhi; Mycobacterium e.g. M. tuberculosis, M. leprae;Clostridium, e.g. C. botulinum, C. tetani, C. difficile, C. perfringens;Cornyebacterium, e.g. C. diphtheriae; Streptococcus, S. pyogenes, S.pneumoniae; Staphylococcus, e.g. S. aureus; Haemophilus, e.g. H.influenzae; Neisseria, e.g. N. meningitidis, N. gonorrhoeae; Yersinia,e.g. Y. lamblia, Y. pestis, Pseudomonas, e.g. P. aeruginosa, P. putida;Chlamydia, e.g. C. trachomatis; Bordetella, e.g. B. pertussis;Treponema, e.g. T. palladium; B. anthracis, Y. pestis, Brucella spp., F.tularensis, B. mallei, B. pseudomallei, B. mallei, B. pseudomallei, C.botulinum, Salmonella spp., SEB V. cholerae toxin B, E. coli O157:H7,Listeria spp., Trichosporon beigelii, Rhodotorula species, Hansenulaanomala, Enterobacter sp., Klebsiella sp., Listeria sp., Mycoplasma sp.and the like.

In some aspects, the antibodies are directed against viral infections;these viruses include, but are not limited to, includingorthomyxoviruses, (e.g. influenza virus), paramyxoviruses (e.grespiratory syncytial virus, mumps virus, measles virus), adenoviruses,rhinoviruses, coronaviruses, reoviruses, togaviruses (e.g. rubellavirus), parvoviruses, poxviruses (e.g. variola virus, vaccinia virus),enteroviruses (e.g. poliovirus, coxsackievirus), hepatitis viruses(including A, B and C), herpesviruses (e.g. Herpes simplex virus,varicella-zoster virus, cytomegalovirus, Epstein-Barr virus),rotaviruses, Norwalk viruses, hantavirus, arenavirus, rhabdovirus (e.g.rabies virus), retroviruses (including HIV, HTLV-I and -II),papovaviruses (e.g. papillomavirus), polyomaviruses, and picornaviruses,and the like.

Optimized IgG Variant Properties

The present application also provides IgG variants that are optimizedfor a variety of therapeutically relevant properties. An IgG variantthat is engineered or predicted to display one or more optimizedproperties is herein referred to as an “optimized IpG variant”. The mostpreferred properties that may be optimized include but are not limitedto enhanced or reduced affinity for FcRn and increased or decreased invivo half-life. Suitable embodiments include antibodies that exhibitincreased binding affinity to FcRn at lowered pH, such as the pHassociated with endosomes, e.g. pH 6.0, while maintaining the reducedaffinity at higher pH, such as 7.4., to allow increased uptake intoendosomes but normal release rates. Similarly, these antibodies withmodulated FcRn binding may optionally have other desirable properties,such as modulated FcγR binding, such as outlined in U.S. Ser. Nos.11/174,287, 11/124,640, 10/822,231, 10/672,280, 10/379,392, and thepatent application entitled IgG Immunoglobulin variants with optimizedeffector function filed on Oct. 21, 2005 having application Ser. No.11/256,060. That is, optimized properties also include but are notlimited to enhanced or reduced affinity for an FcγR. In one optionalembodiment, the IgG variants are optimized to possess enhanced affinityfor a human activating FcγR, preferably FcγRIIIa in addition to the FcRnbinding profile. In yet another optional alternate embodiment, the IgGvariants are optimized to possess reduced affinity for the humaninhibitory receptor FcγRIIb. That is, particular embodiments embrace theuse of antibodies that show increased binding to FcRn, and increasedbinding to FcγRIIIa. Other embodiments utilize use of antibodies thatshow increased binding to FcRn, and increased binding to FcγRIIIa. Theseembodiments are anticipated to provide IgG polypeptides with enhancedtherapeutic properties in humans, for example enhanced effector functionand greater anti-cancer potency. In an alternate embodiment, the IgGvariants are optimized to have increased or reduced affinity for FcRnand increased or decreased affinity for a human FcγR, including but notlimited to FcγRI, FcγRIIa, FcγRIIb, FcγRIIc, FcγRIIIa, and FcγRIIIbincluding their allelic variations. These embodiments are anticipated toprovide IgG polypeptides with enhanced therapeutic properties in humans,for example increased serum half-life and reduced effector function. Inother embodiments, IgG variants provide enhanced affinity for FcRn andenhanced affinity for one or more FcγRs, yet reduced affinity for one ormore other FcγRs. For example, an IgG variant may have enhanced bindingto FcRn and FcγRIIIa, yet reduced binding to FcγRIIb. Alternately, anIgG variant may have reduced binding to FcRn and to FcγR's. In anotherembodiment, an IgG variant may have reduced affinity for FcRn andenhanced affinity for FcγRIIb, yet reduced affinity to one or moreactivating FcγRs. In yet another embodiment, an IgG variant may haveincreased serum half-life and reduced effector functions.

Preferred embodiments comprise optimization of binding to a human FcRnand FcγR, however in alternate embodiments the IgG variants possessenhanced or reduced affinity for FcRn and FcγR from nonhuman organisms,including but not limited to rodents and non-human primates. IgGvariants that are optimized for binding to a nonhuman FcRn may find usein experimentation. For example, mouse models are available for avariety of diseases that enable testing of properties such as efficacy,toxicity, and pharmacokinetics for a given drug candidate. As is knownin the art, cancer cells can be grafted or injected into mice to mimic ahuman cancer, a process referred to as xenografting. Testing of IgGvariants that comprise IgG variants that are optimized for FcRn mayprovide valuable information with regard to the clearancecharacteristics of the protein, its mechanism of clearance, and thelike. The IgG variants may also be optimized for enhanced functionalityand/or solution properties in aglycosylated form. The Fc ligands includebut are not limited to FcRn, FcγRs, C1q, and proteins A and G, and maybe from any source including but not limited to human, mouse, rat,rabbit, or monkey, preferably human. In an alternately preferredembodiment, the IgG variants are optimized to be more stable and/or moresoluble than the aglycosylated form of the parent IgG variant.

IgG variants can include modifications that modulate interaction with Fcligands other than FcRn and FcγRs, including but not limited tocomplement proteins, and Fc receptor homologs (FcRHs). FcRHs include butare not limited to FcRH1, FcRH2, FcRH3, FcRH4, FcRH5, and FcRH6 (Daviset al., 2002, Immunol. Reviews 190:123-136, entirely incorporated byreference).

Preferably, the Fc ligand specificity of the IgG variant will determineits therapeutic utility. The utility of a given IgG variant fortherapeutic purposes will depend on the epitope or form of the targetantigen and the disease or indication being treated. For most targetsand indications, enhanced FcRn binding may be preferable as the enhancedFcRn binding may result in an increase in serum half-life. Longer serumhalf-lives allow less frequent dosing or lower dosing of thetherapeutic. This is particularly preferable when the therapeutic agentis given in response to an indication that requires repeatedadministration. For some targets and indications, decreased FcRnaffinity may be preferable. This may be particularly preferable when avariant Fc with increased clearance or decreased serum half-life isdesired, for example in Fc polypeptides used as imaging agents orradio-therapeutics.

IgG variants may be used that comprise IgG variants that provideenhanced affinity for FcRn with enhanced activating FcγRs and/or reducedaffinity for inhibitory FcγRs. For some targets and indications, it maybe further beneficial to utilize IgG variants that provide differentialselectivity for different activating FcγRs; for example, in some casesenhanced binding to FcγRIIa and FcγRIIIa may be desired, but not FcγRI,whereas in other cases, enhanced binding only to FcγRIIa may bepreferred. For certain targets and indications, it may be preferable toutilize IgG variants that alter FcRn binding and enhance bothFcγR-mediated and complement-mediated effector functions, whereas forother cases it may be advantageous to utilize IgG variants that enhanceFcRn binding, or serum half-life, and either FcγR-mediated orcomplement-mediated effector functions. For some targets or cancerindications, it may be advantageous to reduce or ablate one or moreeffector functions, for example by knocking out binding to C1q, one ormore FcγR's, FcRn, or one or more other Fc ligands. For other targetsand indications, it may be preferable to utilize IgG variants thatprovide enhanced binding to the inhibitory FcγRIIb, yet WT level,reduced, or ablated binding to activating FcγRs. This may beparticularly useful, for example, when the goal of an IgG variant is toinhibit inflammation or auto-immune disease, or modulate the immunesystem in some way. Because auto-immune diseases are generallylong-lasting and treatment is given in repeated dosing, their treatmentwith Fc variants with increased half-life from increased FcRn isparticularly preferred.

Modification may be made to improve the IgG stability, solubility,function, or clinical use. In a preferred embodiment, the IgG variantscan include modifications to reduce immunogenicity in humans. In a mostpreferred embodiment, the immunogenicity of an IgG variant is reducedusing a method described in U.S. Ser. No. 11/004,590, entirelyincorporated by reference. In alternate embodiments, the IgG variantsare humanized (Clark, 2000, Immunol Today 21:397-402, entirelyincorporated by reference).

The IgG variants can include modifications that reduce immunogenicity.Modifications to reduce immunogenicity can include modifications thatreduce binding of processed peptides derived from the parent sequence toMHC proteins. For example, amino acid modifications would be engineeredsuch that there are no or a minimal number of immune epitopes that arepredicted to bind, with high affinity, to any prevalent MHC alleles.Several methods of identifying MHC-binding epitopes in protein sequencesare known in the art and may be used to score epitopes in an IgGvariant. See for example WO 98/52976; WO 02/079232; WO 00/3317; U.S.Ser. No. 09/903,378; U.S. Ser. No. 10/039,170; U.S. Ser. No. 60/222,697;U.S. Ser. No. 10/754,296; PCT WO 01/21823; and PCT WO 02/00165; Mallios,1999, Bioinformatics 15: 432-439; Mallios, 2001, Bioinformatics 17:942-948; Sturniolo et al., 1999, Nature Biotech. 17: 555-561; WO98/59244; WO 02/069232; WO 02/77187; Marshall et al., 1995, J. Immunol.154: 5927-5933; and Hammer et al., 1994, J. Exp. Med. 180: 2353-2358,all entirely incorporated by reference. Sequence-based information canbe used to determine a binding score for a given peptide-MHC interaction(see for example Mallios, 1999, Bioinformatics 15: 432-439; Mallios,2001, Bioinformatics 17: p942-948; Sturniolo et. al., 1999, NatureBiotech. 17: 555-561, all entirely incorporated by reference).

Engineering IgG Variants

Variants of the present invention may be designed by various means. Thevariants, as described herein, may be insertions, deletions,substitutions, other modifications, or combinations of these and otherchanges. A particularly novel embodiment of the present invention is thedesign of insertions and deletions that either improve or reduce thebinding of an Fc polypeptide to an Fc ligand. As disclosed herein,insertions or deletions may be made that increase or decrease theaffinity of the Fc polypeptide for FcRn. Insertions and deletions may bedesigned by rational approaches or by approaches that include the use orrandom components, such as random or semi-random library creation orscreening. In an alternative embodiment, substitutions are disclosedthat increase or decrease the affinity of the Fc polypeptide for FcRn.

Backbone Modifications: Insertions and Deletions

Variant Fc polypeptides may be created by substituting a variant aminoacid in place of the parent amino acid at a position in the Fcpolypeptide. By substituting one or more amino acids for variant aminoacids in the Fc polypeptide, the side chains at those positions arealtered. Most useful substitutions modify the Fc properties by alteringthe Fc side chains. The substituted side chains may interact directly orindirectly with an Fc binding partner that is associated with an Fcfunction or property. The at least one substitution alters the covalentstructure of one or more side chains of the parent Fc polypeptide.

Alternatively, variant Fc polypeptides may be created that change thecovalent structure of the backbone of the parent Fc polypeptide. Thebackbone atoms in proteins are the peptide nitrogen, the alpha carbon,the carbonyl or peptide carbon and the carbonyl oxygen. Changing thecovalent structure of the backbone provides additional methods ofaltering the properties of the Fc polypeptides. The covalent structureof the Fc backbone may be altered by the addition of atoms into thebackbone, e.g. by inserting one or more amino acids, or the subtractionof atoms from the backbone, e.g. by deleting one or more amino acids.The covalent structure of the backbone may also be altered by changingindividual atoms of the backbone to other atoms (Deechongkit et al., JAm Chem. Soc. 2004. 126(51):16762-71, entirely incorporated byreference). As is known in the art and is illustrated herein, insertionsor deletions of amino acids in Fc polypeptides may be done by insertingor deleting the corresponding nucleotides in the DNA encoding the Fcpolypeptide. Alternatively, as is known in the art, insertions ordeletions of amino acids may be done during synthesis of Fcpolypeptides.

The design of insertions or deletions of amino acids that alter theinteraction of the Fc polypeptide with one or more binding partners(e.g. FcgammaR's, FcRn, C1q) may be done by considering the structure ofthe complex of the Fc polypeptide and its binding partner. In a lesspreferred embodiment, the design may be done by considering thestructure of the Fc polypeptide and information about the Fc regioninvolved in binding the binding partner. This information may beobtained by mutagenesis experiments, phage display experiments, homologycomparisons, computer modeling or other means.

Preferred positions in the amino acid sequence for insertions ordeletions that affect the Fc binding interactions, but do not affect theoverall structure, stability, expression or use of the Fc polypeptide,are in loops that are involved in the Fc/Fc-binding partnerinteractions. To alter FcRn binding to the Fc polypeptide, positions244-257, 279-284, 307-317, 383-390, and 428-435 are preferred looplocations for insertions or deletions (numbering from EU index of Kabatet al., Burmeister et al., 1994, Nature, 372:379-383; Martin et al.,2001, Mol Cell 7:867-877, all entirely incorporated by reference). Toalter the Fcgamma receptor binding to the Fc polypeptide, positions229-239, 266-273, 294-299, and 324-331 are preferred loop locations forinsertions or deletions (numbering from EU index of Kabat et al., PDBcode 1E4K.pdb Sondermann et al. Nature. 2000 406:267, all entirelyincorporated by reference). Loops are regions of the polypeptide notinvolved in alpha helical or beta sheet structure. Loops positions arepositions that are not in either alpha helical or beta sheet structures(van Holde, Johnson and Ho. Principles of Physical Biochemistry.Prentice Hall, N.J. 1998, Chapter 1 pp 2-67, entirely incorporated byreference). Loop positions are preferred because the backbone atoms aretypically more flexible and less likely involved in hydrogen bondscompared to the backbone atoms of alpha helices and beta sheets.Therefore, the lengthening or shortening of a loop due to an insertionor deletion of one or more amino acids is less likely to lead to large,disruptive changes to the Fc polypeptide, including stability,expression or other problems.

Insertions and deletions may be used to alter the length of thepolypeptide. For example, in loop regions, altering the loop lengthresults in altered flexibility and conformational entropy of the loop.Insertions in a loop will generally increase the conformational entropyof the loop, which may be defined as Boltzman's constant multiplied bythe natural logarithm of the number of possible conformations (vanHolde, Johnson and Ho. Principles of Physical Biochemistry. PrenticeHall, N.J. 1998, pp 78, entirely incorporated by reference). Byinserting at least one amino acid into a polypeptide, the total numberof conformations available to the polypeptide increases. Theseadditional conformations may be beneficial for forming favorableFc/Fc-binding partner interactions because the Fc polypeptide may useone of the additional conformations in binding the Fc-binding protein.In this case, the insertion may lead to stronger Fc/Fc-binding partnerinteractions. If the additional conformations are not used in thebinding interface, then the insertion may lead to weaker Fc/Fc-bindingpartner interactions, because the additional conformations would competewith the binding-competent conformation. Similarly, deletion of apolypeptide segment may also lead to either stronger or weaker Fc/Fcbinding-partner interactions. If deletion of a segment, which reducesthe possible number of backbone conformations, removes thebinding-competent conformation, then the deletion may lead to weakerFc/Fc-binding partner interactions. If the deletion does not remove thebinding-competent conformation, then the deletion may lead to strongerFc/Fc-binding partner interactions because the deletion may removeconformations that compete with the binding-competent conformation.

Insertions and deletions may be used to alter the positions andorientations of the amino acids in the Fc polypeptide. Becauseinsertions and deletions cause a change in the covalent structure of thebackbone, they necessarily cause a change in the positions of thebackbone atoms. FIG. 7 compares the backbone positions at some loopsegments, marked L1 to L4, in three different backbones. The referencebackbone structure contains four loop segments, whereas the deletionbackbone lacks segment L1 and the insertion segment comprises anadditional segment before, ie, N-terminal to, segment L1. Deletions andinsertions cause the largest change in the backbone structure near thesite of the insertion or deletion. By deleting a segment near theN-terminal end of the loop, e.g. segment L1, the loop shortens and theremaining segments shift their position closer to the loop N-terminus.This has the effect of moving the L2 segment toward the prior locationof the L1 segment and toward the loop N-terminus. This change inposition of the L2 segment toward the L1 segment may strengthen thebinding of the Fc/Fc-binding partner complex and is preferred when thereis prior information suggesting that the amino acid or amino acidslocated in L2 make favorable interactions with the Fc-binding partner,when located in L1. For example, if L2 contains alanine and tyrosine andsubstitution of two L1 amino acids for alanine and tyrosine previouslylead to an Fc variant with increased binding, then deletion of L1 maycreate an Fc variant with increased affinity for the Fc-binding partner.

Similarly, an insertion of a polypeptide segment into an Fc polypeptideat the N-terminal side of a loop causes the positions of the loopsegments to be shifted toward the C-terminal side of the loop. In FIG.7, an insertion of one or more amino acids before, i.e. N-terminally to,segment L1 alters the backbone conformation including a shift of the L1segment toward the C-terminal end of the loop. This type of insertion ispreferred when the amino acids located in segment L1 are known to makefavorable interactions when located in the L2 positions, as theinsertion may lead to stronger Fc/Fc-binding partner interactions. Ifweaker Fc/Fc-binding partner interactions are desired, then theinsertion may be used to shift unfavorable amino acid into a newposition. The inserted, deleted and reference segments (L1 to L4 in FIG.7) may be one or more than one amino acid in the Fc polypeptide.

Alternatively, insertions or deletions may be used at the C-terminal endof loops in a manner analogous to the insertions or deletions at theN-terminal end of loops. Insertions at the loop C-terminus may lead to amovement of the positions N-terminal of the insertion toward the loopN-terminus. Deletions at the loop C-terminus may lead to a movement ofthe positions N-terminal of the deletion toward the loop C-terminus. Thechoice of using an insertion or deletion at the N-terminal or C-terminalend of the loop is based on the amino acids located in the loop, thedesire for increased or decreased Fc/Fc-binding partner affinity, andthe positional shift desired.

Insertions or deletions may be used in any region of an Fc polypeptide,including the loops, the alpha helical, and the beta sheet regions.Preferred locations for insertions and deletions include loop regions,which are those that are not alpha helical or beta sheet regions. Loopsare preferred because they generally accept alterations in the backbonebetter than alpha helixes or beta sheets. The particularly preferredlocations for insertions or deletions that result in strongerprotein/protein interactions are at the N-terminal or C-terminal edgesof a loop. If the loop side chains are involve in the Fc/Fc-bindingpartner interactions, then insertions or deletion at the edges are lesslikely to lead to strongly detrimental changes in the bindinginteractions. Deletions within the exact center of the loop are morelikely to remove important residues in the Fc/Fc-binding partnerinterface and insertions within the exact center of the loop are morelikely to create unfavorable interactions in the Fc/Fc-binding partnerinterface. The number of residues deleted or inserted may be determinedby the size of the backbone change desired with insertions or deletionsof 15 or less residues being preferred, insertions or deletions of 10 orless residues being more preferred, and insertions or deletions of 5 orless residues being most preferred.

Once the position and size of an Fc deletion variant is designed, theentire polypeptide sequence is completely determined and the polypeptidemay be constructed by methods known in the art.

Fc insertion variants, however, have the additional step of designingthe sequence of the at least one amino acid to be inserted. Insertionsof polar residues, including Ser, Thr, Asn, Gln, Ala, Gly, His, arepreferred at positions expected to be exposed in the Fc polypeptide. Thesmaller amino acids, including Ser, Thr, and Ala, are particularlypreferred as the small size is less likely to sterically interfere withthe Fc/Fc-binding partner interactions. Ser and Thr also have thecapability to hydrogen bond with atoms on the Fc-binding partner.

Insertions also have the added flexibility that the inserted polypeptidemay be designed to make favorable interactions with the Fc-bindingpartner as would be desire when stronger Fc/Fc-binding partner bindingis desired. The length of the backbone insertion may be determined bymodeling the variant backbone with a simple, generic sequence to beinserted. For example, polyserine, polyglycine or polyalanine insertionsof different lengths may be constructed and modeled. Modeling may bedone by a variety of methods, including homology modeling based on knownthree-dimensional structures of homologues comprising the insertion, andby computer modeling including MODELLER (M. A. Marti-Renom et al. Annu.Rev. Biophys. Biomol. Struct. 29, 291-325, 2000) and ROSETTA (Kuhlman etal. (2003). Science 302, 1364-8), both entirely incorporated byreference. Typically, various backbone conformations are initiallygenerated and the final backbone structure may be determined after theidentities of the side chain are established. The side chains may bedesigned by PDA® algorithms (U.S. Pat. Nos. 6,188,965; 6,269,312;6,403,312; 6,801,861; 6,804,611; 6,792,356, 6,950,754, and U.S. Ser. No.09/782,004; 09/927,790; 10/101,499; 10/666,307; 10/666,311; 10/218,102,all entirely incorporated by reference).

Insertions and deletions may be made to alter the binding of Fcpolypeptides to FcgammaR in an analogous manner to the described methodto alter FcRn-binding properties. Fc domains bind to the FcgammaR at theposition indicated in FIG. 1. Structures of the Fc/FcgammaR complex,including PDB codes 1T89 and 1IIS (Radaev S et al. J. Biol. Chem. v276,p. 16469-16477 entirely incorporated by reference), demonstrate theinteracting residues and loops between the two structures. Mutagenesisresults such as those found in U.S. Ser. No. 11/124,620 and U.S. Pat.No. 6,737,056, both entirely incorporated by reference) all have utilityin determined appropriate shifts of backbone positioning.

Insertions and deletions may be designed in any polypeptide besides Fcpolypeptides by the methods described herein. For example, insertions ordeletions in the TNF superfamily member, APRIL, may be designed with theaid of its three-dimensional structure (PDB code 1XU1.pdb, Hymowitz, etal. (2005) J. Biol. Chem. 280:7218, entirely incorporated by reference).Insertions or deletions may be designed to increase APRIL binding to itsreceptor, TACI. The loop residues preferred as insertion or deletionsites are residues Ser118-Val124, Asp164-Phe167, Pro192-Ala198,Pro221-Lys226. These loops interact with TACI in the APRIL/TACI complexand mediate binding.

Polypeptides Incorporating Variants

The IgG variants can be based on human IgG sequences, and thus human IgGsequences are used as the “base” sequences against which other sequencesare compared, including but not limited to sequences from otherorganisms, for example rodent and primate sequences. IgG variants mayalso comprise sequences from other immunoglobulin classes such as IgA,IgE, IgD, IgM, and the like. It is contemplated that, although the IgGvariants are engineered in the context of one parent IgG, the variantsmay be engineered in or “transferred” to the context of another, secondparent IgG. This is done by determining the “equivalent” or“corresponding” residues and substitutions between the first and secondIgG, typically based on sequence or structural homology between thesequences of the IgGs. In order to establish homology, the amino acidsequence of a first IgG outlined herein is directly compared to thesequence of a second IgG. After aligning the sequences, using one ormore of the homology alignment programs known in the art (for exampleusing conserved residues as between species), allowing for necessaryinsertions and deletions in order to maintain alignment (i.e., avoidingthe elimination of conserved residues through arbitrary deletion andinsertion), the residues equivalent to particular amino acids in theprimary sequence of the first IgG variant are defined. Alignment ofconserved residues preferably should conserve 100% of such residues.However, alignment of greater than 75% or as little as 50% of conservedresidues is also adequate to define equivalent residues. Equivalentresidues may also be defined by determining structural homology betweena first and second IgG that is at the level of tertiary structure forIgGs whose structures have been determined. In this case, equivalentresidues are defined as those for which the atomic coordinates of two ormore of the main chain atoms of a particular amino acid residue of theparent or precursor (N on N, CA on CA, C on C and O on O) are within0.13 nm and preferably 0.1 nm after alignment. Alignment is achievedafter the best model has been oriented and positioned to give themaximum overlap of atomic coordinates of non-hydrogen protein atoms ofthe proteins. Regardless of how equivalent or corresponding residues aredetermined, and regardless of the identity of the parent IgG in whichthe IgGs are made, what is meant to be conveyed is that the IgG variantsdiscovered by can be engineered into any second parent IgG that hassignificant sequence or structural homology with the IgG variant. Thusfor example, if a variant antibody is generated wherein the parentantibody is human IgG1, by using the methods described above or othermethods for determining equivalent residues, the variant antibody may beengineered in another IgG1 parent antibody that binds a differentantigen, a human IgG2 parent antibody, a human IgA parent antibody, amouse IgG2a or IgG2b parent antibody, and the like. Again, as describedabove, the context of the parent IgG variant does not affect the abilityto transfer the IgG variants to other parent IgGs.

Methods for engineering, producing, and screening IgG variants areprovided. The described methods are not meant to constrain to anyparticular application or theory of operation. Rather, the providedmethods are meant to illustrate generally that one or more IgG variantsmay be engineered, produced, and screened experimentally to obtain IgGvariants with optimized effector function. A variety of methods aredescribed for designing, producing, and testing antibody and proteinvariants in U.S. Ser. No. 10/754,296, and U.S. Ser. No. 10/672,280, bothentirely incorporated by reference.

A variety of protein engineering methods may be used to design IgGvariants with optimized effector function. In one embodiment, astructure-based engineering method may be used, wherein availablestructural information is used to guide substitutions, insertions ordeletions. In a preferred embodiment, a computational screening methodmay be used, wherein substitutions are designed based on their energeticfitness in computational calculations. See for example U.S. Ser. No.10/754,296 and U.S. Ser. No. 10/672,280, and references cited therein,all entirely incorporated by reference.

An alignment of sequences may be used to guide substitutions at theidentified positions. One skilled in the art will appreciate that theuse of sequence information may curb the introduction of substitutionsthat are potentially deleterious to protein structure. The source of thesequences may vary widely, and include one or more of the knowndatabases, including but not limited to the Kabat database (NorthwesternUniversity); Johnson & Wu, 2001, Nucleic Acids Res. 29:205-206; Johnson& Wu, 2000, Nucleic Acids Res. 28:214-218), the IMGT database (IMGT, theinternational ImMunoGeneTics information System®; Lefranc et al., 1999,Nucleic Acids Res. 27:209-212; Ruiz et al., 2000 Nucleic Acids Res.28:219-221; Lefranc et al., 2001, Nucleic Acids Res. 29:207-209; Lefrancet al., 2003, Nucleic Acids Res. 31:307-310), and VBASE, all entirelyincorporated by reference. Antibody sequence information can beobtained, compiled, and/or generated from sequence alignments ofgermline sequences or sequences of naturally occurring antibodies fromany organism, including but not limited to mammals. One skilled in theart will appreciate that the use of sequences that are human orsubstantially human may further have the advantage of being lessimmunogenic when administered to a human. Other databases which are moregeneral nucleic acid or protein databases, i.e. not particular toantibodies, include but are not limited to SwissProt, GenBank Entrez,and EMBL Nucleotide Sequence Database. Aligned sequences can include VH,VL, CH, and/or CL sequences. There are numerous sequence-based alignmentprograms and methods known in the art, and all of these find use in thegeneration of sequence alignments.

Alternatively, random or semi-random mutagenesis methods may be used tomake amino acid modifications at the desired positions. In these casespositions are chosen randomly, or amino acid changes are made usingsimplistic rules. For example all residues may be mutated to alanine,referred to as alanine scanning. Such methods may be coupled with moresophisticated engineering approaches that employ selection methods toscreen higher levels of sequence diversity. As is well known in the art,there are a variety of selection technologies that may be used for suchapproaches, including, for example, display technologies such as phagedisplay, ribosome display, cell surface display, and the like, asdescribed below.

Methods for production and screening of IgG variants are well known inthe art. General methods for antibody molecular biology, expression,purification, and screening are described in Antibody Engineering,edited by Duebel & Kontermann, Springer-Verlag, Heidelberg, 2001; andHayhurst & Georgiou, 2001, Curr Opin Chem Biol 5:683-689; Maynard &Georgiou, 2000, Annu Rev Biomed Eng 2:339-76. Also see the methodsdescribed in U.S. Ser. No. 10/754,296; U.S. Ser. No. 10/672,280; andU.S. Ser. Nos. 10/822,231; and 11/124,620, all entirely incorporated byreference.

Preferred variants of the present invention include those found in FIG.8. Alternatively preferred variants of the present invention includethose found in FIG. 9. Additionally alternatively preferred variants ofthe present invention include those found in FIG. 10. These variantshave shown increased binding to the Fc receptor, FcRn, as illustrated inthe examples.

Making IgG Variants

The IgG variants can be made by any method known in the art. In oneembodiment, the IgG variant sequences are used to create nucleic acidsthat encode the member sequences, and that may then be cloned into hostcells, expressed and assayed, if desired. These practices are carriedout using well-known procedures, and a variety of methods that may finduse in are described in Molecular Cloning—A Laboratory Manual, 3^(rd)Ed. (Maniatis, Cold Spring Harbor Laboratory Press, New York, 2001), andCurrent Protocols in Molecular Biology (John Wiley & Sons), bothentirely incorporated by reference. The nucleic acids that encode theIgG variants may be incorporated into an expression vector in order toexpress the protein. Expression vectors typically include a proteinoperably linked, that is, placed in a functional relationship, withcontrol or regulatory sequences, selectable markers, any fusionpartners, and/or additional elements. The IgG variants may be producedby culturing a host cell transformed with nucleic acid, preferably anexpression vector, containing nucleic acid encoding the IgG variants,under the appropriate conditions to induce or cause expression of theprotein. A wide variety of appropriate host cells may be used, includingbut not limited to mammalian cells, bacteria, insect cells, and yeast.For example, a variety of cell lines that may find use are described inthe ATCC cell line catalog, available from the American Type CultureCollection, entirely incorporated by reference. The methods ofintroducing exogenous nucleic acid into host cells are well known in theart, and will vary with the host cell used.

In a preferred embodiment, IgG variants are purified or isolated afterexpression. Antibodies may be isolated or purified in a variety of waysknown to those skilled in the art. Standard purification methods includechromatographic techniques, electrophoretic, immunological,precipitation, dialysis, filtration, concentration, and chromatofocusingtechniques. As is well known in the art, a variety of natural proteinsbind antibodies, for example bacterial proteins A, G, and L, and theseproteins may find use in purification. Often, purification may beenabled by a particular fusion partner. For example, proteins may bepurified using glutathione resin if a GST fusion is employed, Ni⁺²affinity chromatography if a His-tag is employed, or immobilizedanti-flag antibody if a flag-tag is used. For general guidance insuitable purification techniques, see Antibody Purification: Principlesand Practice, 3^(rd) Ed., Scopes, Springer-Verlag, N.Y., 1994, entirelyincorporated by reference.

Screening IgG Variants

Fc variants may be screened using a variety of methods, including butnot limited to those that use in vitro assays, in vivo and cell-basedassays, and selection technologies. Automation and high-throughputscreening technologies may be utilized in the screening procedures.Screening may employ the use of a fusion partner or label, for examplean immune label, isotopic label, or small molecule label such as afluorescent or colorimetric dye.

In a preferred embodiment, the functional and/or biophysical propertiesof Fc variants are screened in an in vitro assay. In a preferredembodiment, the protein is screened for functionality, for example itsability to catalyze a reaction or its binding affinity to its target.

As is known in the art, subsets of screening methods are those thatselect for favorable members of a library. The methods are hereinreferred to as “selection methods”, and these methods find use in thepresent invention for screening Fc variants. When protein libraries arescreened using a selection method, only those members of a library thatare favorable, that is which meet some selection criteria, arepropagated, isolated, and/or observed. A variety of selection methodsare known in the art that may find use in the present invention forscreening protein libraries. Other selection methods that may find usein the present invention include methods that do not rely on display,such as in vivo methods. A subset of selection methods referred to as“directed evolution” methods are those that include the mating orbreading of favorable sequences during selection, sometimes with theincorporation of new mutations.

In a preferred embodiment, Fc variants are screened using one or morecell-based or in vivo assays. For such assays, purified or unpurifiedproteins are typically added exogenously such that cells are exposed toindividual variants or pools of variants belonging to a library. Theseassays are typically, but not always, based on the function of the Fcpolypeptide; that is, the ability of the Fc polypeptide to bind to itstarget and mediate some biochemical event, for example effectorfunction, ligand/receptor binding inhibition, apoptosis, and the like.Such assays often involve monitoring the response of cells to the IgG,for example cell survival, cell death, change in cellular morphology, ortranscriptional activation such as cellular expression of a natural geneor reporter gene. For example, such assays may measure the ability of Fcvariants to elicit ADCC, ADCP, or CDC. For some assays additional cellsor components, that is in addition to the target cells, may need to beadded, for example serum complement, or effector cells such asperipheral blood monocytes (PBMCs), NK cells, macrophages, and the like.Such additional cells may be from any organism, preferably humans, mice,rat, rabbit, and monkey. Antibodies may cause apoptosis of certain celllines expressing the target, or they may mediate attack on target cellsby immune cells which have been added to the assay. Methods formonitoring cell death or viability are known in the art, and include theuse of dyes, immunochemical, cytochemical, and radioactive reagents.Transcriptional activation may also serve as a method for assayingfunction in cell-based assays. Alternatively, cell-based screens areperformed using cells that have been transformed or transfected withnucleic acids encoding the variants. That is, Fc variants are not addedexogenously to the cells.

The biological properties of the IgG variants may be characterized incell, tissue, and whole organism experiments. As is known in the art,drugs are often tested in animals, including but not limited to mice,rats, rabbits, dogs, cats, pigs, and monkeys, in order to measure adrug's efficacy for treatment against a disease or disease model, or tomeasure a drug's pharmacokinetics, toxicity, and other properties. Theanimals may be referred to as disease models. Therapeutics are oftentested in mice, including but not limited to nude mice, SCID mice,xenograft mice, and transgenic mice (including knockins and knockouts).Such experimentation may provide meaningful data for determination ofthe potential of the protein to be used as a therapeutic. Any organism,preferably mammals, may be used for testing. For example because oftheir genetic similarity to humans, monkeys can be suitable therapeuticmodels, and thus may be used to test the efficacy, toxicity,pharmacokinetics, or other property of the IgGs. Tests of the in humansare ultimately required for approval as drugs, and thus of course theseexperiments are contemplated. Thus the IgGs may be tested in humans todetermine their therapeutic efficacy, toxicity, immunogenicity,pharmacokinetics, and/or other clinical properties.

Methods of Using IgG Variants

The IgG variants may find use in a wide range of products. In oneembodiment the IgG variant is a therapeutic, a diagnostic, or a researchreagent, preferably a therapeutic. The IgG variant may find use in anantibody composition that is monoclonal or polyclonal. In a preferredembodiment, the IgG variants are used to kill target cells that bear thetarget antigen, for example cancer cells. In an alternate embodiment,the IgG variants are used to block, antagonize or agonize the targetantigen, for example for antagonizing a cytokine or cytokine receptor.In an alternately preferred embodiment, the IgG variants are used toblock, antagonize or agonize the target antigen and kill the targetcells that bear the target antigen.

The IgG variants may be used for various therapeutic purposes. In apreferred embodiment, an antibody comprising the IgG variant isadministered to a patient to treat an antibody-related disorder. A“patient” for the purposes includes humans and other animals, preferablymammals and most preferably humans. By “antibody related disorder” or“antibody responsive disorder” or “condition” or “disease” herein aremeant a disorder that may be ameliorated by the administration of apharmaceutical composition comprising an IgG variant. Antibody relateddisorders include but are not limited to autoimmune diseases,immunological diseases, infectious diseases, inflammatory diseases,neurological diseases, and oncological and neoplastic diseases includingcancer. By “cancer” and “cancerous” herein refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto carcinoma, lymphoma, blastoma, sarcoma (including liposarcoma),neuroendocrine tumors, mesothelioma, schwanoma, meningioma,adenocarcinoma, melanoma, and leukemia and lymphoid malignancies.

In one embodiment, an IgG variant is the only therapeutically activeagent administered to a patient. Alternatively, the IgG variant isadministered in combination with one or more other therapeutic agents,including but not limited to cytotoxic agents, chemotherapeutic agents,cytokines, growth inhibitory agents, anti-hormonal agents, kinaseinhibitors, anti-angiogenic agents, cardioprotectants, or othertherapeutic agents. The IgG variants may be administered concomitantlywith one or more other therapeutic regimens. For example, an IgG variantmay be administered to the patient along with chemotherapy, radiationtherapy, or both chemotherapy and radiation therapy. In one embodiment,the IgG variant may be administered in conjunction with one or moreantibodies, which may or may not be an IgG variant. In accordance withanother embodiment, the IgG variant and one or more other anti-cancertherapies are employed to treat cancer cells ex vivo. It is contemplatedthat such ex vivo treatment may be useful in bone marrow transplantationand particularly, autologous bone marrow transplantation. It is ofcourse contemplated that the IgG variants can be employed in combinationwith still other therapeutic techniques such as surgery.

A variety of other therapeutic agents may find use for administrationwith the IgG variants. In one embodiment, the IgG is administered withan anti-angiogenic agent. By “anti-angiogenic agent” as used herein ismeant a compound that blocks, or interferes to some degree, thedevelopment of blood vessels. The anti-angiogenic factor may, forinstance, be a small molecule or a protein, for example an antibody, Fcfusion, or cytokine, that binds to a growth factor or growth factorreceptor involved in promoting angiogenesis. The preferredanti-angiogenic factor herein is an antibody that binds to VascularEndothelial Growth Factor (VEGF). In an alternate embodiment, the IgG isadministered with a therapeutic agent that induces or enhances adaptiveimmune response, for example an antibody that targets CTLA-4. In analternate embodiment, the IgG is administered with a tyrosine kinaseinhibitor. By “tyrosine kinase inhibitor” as used herein is meant amolecule that inhibits to some extent tyrosine kinase activity of atyrosine kinase. In an alternate embodiment, the IgG variants areadministered with a cytokine.

Pharmaceutical compositions are contemplated wherein an IgG variant andone or more therapeutically active agents are formulated. Formulationsof the IgG variants are prepared for storage by mixing the IgG havingthe desired degree of purity with optional pharmaceutically acceptablecarriers, excipients or stabilizers (Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed., 1980, entirely incorporated by reference),in the form of lyophilized formulations or aqueous solutions. Theformulations to be used for in vivo administration are preferablysterile. This is readily accomplished by filtration through sterilefiltration membranes or other methods. The IgG variants and othertherapeutically active agents disclosed herein may also be formulated asimmunoliposomes, and/or entrapped in microcapsules.

The concentration of the therapeutically active IgG variant in theformulation may vary from about 0.1 to 100% by weight. In a preferredembodiment, the concentration of the IgG is in the range of 0.003 to 1.0molar. In order to treat a patient, a therapeutically effective dose ofthe IgG variant may be administered. By “therapeutically effective dose”herein is meant a dose that produces the effects for which it isadministered. The exact dose will depend on the purpose of thetreatment, and will be ascertainable by one skilled in the art usingknown techniques. Dosages may range from 0.01 to 100 mg/kg of bodyweight or greater, for example 0.01, 0.1, 1.0, 10, or 50 mg/kg of bodyweight, with 1 to 10 mg/kg being preferred. As is known in the art,adjustments for protein degradation, systemic versus localized delivery,and rate of new protease synthesis, as well as the age, body weight,general health, sex, diet, time of administration, drug interaction andthe severity of the condition may be necessary, and will beascertainable with routine experimentation by those skilled in the art.

Administration of the pharmaceutical composition comprising an IgGvariant, preferably in the form of a sterile aqueous solution, may bedone in a variety of ways, including, but not limited to, orally,subcutaneously, intravenously, parenterally, intranasally,intraotically, intraocularly, rectally, vaginally, transdermally,topically (e.g., gels, salves, lotions, creams, etc.),intraperitoneally, intramuscularly, intrapulmonary (e.g., AERx®inhalable technology commercially available from Aradigm, or Inhance®pulmonary delivery system commercially available from NektarTherapeutics, etc.). Therapeutic described herein may be administeredwith other therapeutics concomitantly, i.e., the therapeutics describedherein may be co-administered with other therapies or therapeutics,including for example, small molecules, other biologicals, radiationtherapy, surgery, etc.

EXAMPLES

Examples are provided below to illustrate the present invention. Theseexamples are not meant to constrain the present invention to anyparticular application or theory of operation. For all positionsdiscussed in the present invention, numbering is according to the EUindex as in Kabat (Kabat et al., 1991, Sequences of Proteins ofImmunological Interest, 5th Ed., United States Public Health Service,National Institutes of Health, Bethesda, entirely incorporated byreference). Those skilled in the art of antibodies will appreciate thatthis convention consists of nonsequential numbering in specific regionsof an immunoglobulin sequence, enabling a normalized reference toconserved positions in immunoglobulin families. Accordingly, thepositions of any given immunoglobulin as defined by the EU index willnot necessarily correspond to its sequential sequence.

Example 1 DNA Construction, Expression, and Purification of Fc Variants

Amino acid modifications were engineered in the Fc region of IgGantibodies to improve their affinity for the neonatoal Fc receptor FcRn.Variants were screened in the context of a number of different human IgGconstant chains (FIG. 2), including IgG1, IgG2, and a hybrid IgGsequences that contains the CH1 and upper hinge of IgG1 and the Fcregion of IgG2. It will be appreciated by those skilled in the art thatbecause of the different interactions of the IgG1 and IgG2 Fc regionwith FcγRs and complement, these different parent Fc regions will havedifferent FcγR- and complement-mediated effector function properties.Exemplary sequences of Fc variants in the context of these parent IgGconstant chains are shown in FIG. 3.

Fc variants were engineered in the context of an antibody targetingvascular endothelial factor (VEGF). The heavy and light chain variableregions (VH and VL) are those of a humanized version of the antibodyA4.6.1, also referred to as bevacizumab (Avastin®), which is approvedfor the treatment of a variety of cancers. The amino acid sequences ofthe VH and VL regions of this antibody are shown in FIG. 4.

Genes encoding the heavy and light chains of the anti-VEGF antibodieswere constructed in the mammalian expression vector pTT5. Human IgG1 andIgG2 constant chain genes were obtained from IMAGE clones and subclonedinto the pTT5 vector. The IgG1/2 gene was constructed using PCRmutagenesis. VH and VL genes encoding the anti-VEGF antibodies weresynthesized commercially (Blue Heron Biotechnologies, Bothell Wash.),and subcloned into the vectors encoding the appropriate CL, IgG1, IgG2,and IgG1/2 constant chains. Amino acid modifications were constructedusing site-directed mutagenesis using the QuikChange® site-directedmutagenesis methods (Stratagene, La Jolla Calif.). All DNA was sequencedto confirm the fidelity of the sequences.

Plasmids containing heavy chain gene (VH-Cγ1-Cγ2-Cγ3) wereco-transfected with plasmid containing light chain gene (VL-Cκ) into293E cells using lipofectamine (Invitrogen, Carlsbad Calif.) and grownin FreeStyle 293 media (Invitrogen, Carlsbad Calif.). After 5 days ofgrowth, the antibodies were purified from the culture supernatant byprotein A affinity using the MabSelect resin (GE Healthcare). Antibodyconcentrations were determined by bicinchoninic acid (BCA) assay(Pierce).

Example 2 Fc Variant Antibodies Maintain Binding to Antigen

The fidelity of the expressed variant antibodies was confirmed bydemonstrating that they maintained specificity for antigen. VEGF bindingwas monitored using surface plasmon resonance (SPR, Biacore), performedusing a Biacore 3000 instrument. Recombinant VEGF (VEGF-165, PeproTech,Rocky Hill, N.J.) was adhered to a CM5 chip surface by coupling withN-hydroxysuccinimide/N-ethyl-N′-(-3-dimethylamino-propyl) carbodiimide(NHS/EDC) using standard methods. WT and variant antibodies wereinjected as analytes, and response, measured in resonance units (RU),was acquired. The dissociation phase was too slow to measure trueequilibrium constants, and so relative binding was determined bymeasuring RU's at the end of the association phase, which should beproportional to the protein concentration (which is held constant in theexperiment) and the association rate constant. The data (FIG. 6) showthat the variant anti-VEGF antibodies maintain binding to antigen, incontrast to the negative control anti-Her2 antibody which does not bindVEGF.

Example 3 Measurement of Binding to Human FcRn

Binding of variant antibodies to human FcRn was measured at pH 6.0, thepH at which it is naturally bound in endosomes. Vectors encoding beta 2microglobulin and His-tagged alpha chain genes of FcRn were constructed,co-transfected into 293T cells, and purified using nickelchromatography. Antibody affinity for human FcRn (hFcRn) at pH 6.0 wasmeasured on a Biacore 3000 instrument by coupling human FcRn to a CM5chip surface using standard NHS/EDC chemistry. WT and variant antibodieswere used in the mobile phase at 25-100 nM concentration and responsewas measured in resonance units. Association and dissociation phases atpH 6.0 were acquired, followed by an injection of pH 7.4 buffer tomeasure release of antibody from receptor at the higher pH. A cycle withantibody and buffer only provided baseline response, which wassubtracted from each sample sensorgram.

FIG. 7 shows Biacore sensorgrams for binding of native IgG1 and selectFc variant antibodies to human FcRn at the two relevant pH's. The datashow that wild-type and variant antibodies bind readily to FcRn chip atpH 6.0 and dissociate slowly at that pH, as they would in the endosome,yet release rapidly at pH7.4, as they would upon recycling of endosometo the membrane and exposure to the higher pH of serum.

The FcRn association/dissociation curves did not fit to a simpleLangmuir model, possibly due to the antibody and receptor multi-valencyor chip heterogeneity. Pseudo-Ka values (referred to as Ka*) weredetermined by fitting to a conformational change model with the changein refractive index (RI) fixed at 0 RU. These values for select variantantibodies are plotted in FIG. 8. The relative affinity of each variantas compared to its parent IgG was calculated according to the equationFold=(WT Ka*/Variant Ka*). The relative binding data for all Fc variantsin an IgG1 Fc region are presented in FIG. 9, and binding data forvariants in antibodies with an IgG2 Fc region (constant chains IgG1 andIgG1/2) are presented in FIG. 10. For many variants, the bindingexperiment was repeated multiple times (n), for which folds werecalculated with reference to the WT IgG parent within each particularbinding experiment. Averaging of these data provided a mean and standarddeviation, as presented in FIGS. 9 and 10.

FIGS. 9 and 10 show that a number of engineered variants bind withgreater affinity to human FcRn binding at pH 6.0 relative to WT IgG1.Improvements were heavily dependent on the identity of the substitutionat a given position. For example, using 2-fold as a criteria forimproved binding, a number of mutations at position 434 in IgG2increased affinity (A, S, Y, F, and W), some were neutral (within 2-foldof WT IgG2) (G, H, M, and T), and a number of substitutions reducedaffinity (<0.5 fold) (D, E, K, P, R, and V). Greater binding in thecontext of IgG1 did not necessarily translate to greater binding in IgG2(for example 434T was improved in binding in IgG1 but not IgG2).Moreover, improvements provided by single variants were not alwaysadditive upon combination. FIG. 11 a demonstrates this graphically byplotting the experimental fold FcRn binding by select doublesubstitution variants versus the product of the fold FcRn binding by theindividual single variants that compose them. The straight linerepresents perfect additivity, i.e. the value that would be expected orpredicted from the product of the single substitutions. A number ofdouble variants fall on or close to this line (259I/319I, 259I/428L,319I/428L, and 308F/428L). Several variants are less than additive(319I/308F, 252Y/428L, and 428L/434M). For these variants, particularlyin the case of the latter two (252Y/428L and 428L/434M), the affinityimprovements of the single substitutions would seem to be incompatablewith each other when combined. Surprisingly, the FcRn affinityimprovements of variants 259I/308F and 428L/434S were greater than wouldbe expected from the affinities of their respective singlesubstitutions. These particular single substitutions had unexpectedsynergistic improvements when combined. The difference betweenexperimental affinities and those predicted from the affinities of thesingle variants are plotted in FIG. 11 b, with variants groupedaccording to their composite single variants (259I, 308F, and 319I onthe left, and combinations with 482L on the right). Synergy can bequantitated by calculating the fold of the experimental value relativeto the predicted value, followed by normalization to 1 and conversion toa percentage (% synergy=100×[(experimental fold/predicted fold)−1)].This analysis is plotted in FIG. 11 b, with variants grouped accordingto their composite single variants. This graph highlights again thesynergy of some of the variants, particularly 259I/308F and 428L/434S.FIGS. 11 b and 11 c also emphasize the nonpredictive nature of combiningmany of the best single substitutions from the screen. For example,whereas combination of 428L with 434S and 259I provided synergisticbinding improvements, 252Y or 434M had a negative impact when combinedwith 428L. The dramatic difference between combining 428L with 434Sversus 434M further highlights the importance of the particular aminoacid identity of the substitution at a given position.

Example 4 Testing of Variants in Other Antibody Contexts

Select variants were constructed in the context of antibodies targetingother antigens, including TNF (TNFα), CD25 (TAC), EGFR, and IgE. FIG. 4provides the amino acid sequences of the VH and VL regions of antibodiestargeting these antigens that were used in the invention. The WT and Fcvariant anti-TNF antibodies contain the variable region of the fullyhuman antibody adalimumab (Humira®), currently approved for thetreatment of rheumatoid arthritis (RA), juvenile idiopathic arthritis(JIA), psoriatic arthritis (PsA), ankylosing spondylitis (AS), andCrohn's disease (CD). The WT and Fc variant anti-CD25 antibodies arehumanized versions of the antibody anti-TAC (Junghans et al., 1990,Cancer Research 50:1495-1502), referred to as H1.8/L1 anti-TAC. The WTand Fc variant anti-EGFR antibodies are humanized versions of the murineantibody C225, referred to as H4.42/L3.32 C225. Finally, the WT and Fcvariant anti-IgE antibodies contain the variable region of the humanizedantibody omalizumab (Xolair®), which is approved for the treatment ofallergic asthma.

WT and variant antibodies were constructed, expressed, and purified asdescribed above. Antibodies were tested for binding to human FcRn at pH6.0 by Biacore as described above. The relative binding data of thevariant anti-TNF, -CD25, -EGFR, and -IgE antibodies to human FcRn areprovided in FIG. 12. As can be seen, the variants improve FcRn affinityin the context of antibodies targeting a variety of antigens.

Example 5 Pharmacokinetic Experiments in Human FcRn Knock-in Mice

To test the ability of select variants to improve half-life in vivo,pharmacokinetic experiments were performed in B6 mice that arehomozygous knock-outs for murine FcRn and heterozygous knock-ins ofhuman FcRn (mFcRn^(−/−), hFcRn⁺) (Petkova et al., 2006, Int Immunol18(12):1759-69, entirely incorporated by reference), herein referred toas hFcRn or hFcRn⁺ mice. A single, intravenous tail vein injection ofanti-VEGF antibody (2 mg/kg) was given to groups of 4-7 female micerandomized by body weight (20-30 g range). Blood (˜50 ul) was drawn fromthe orbital plexus at each time point, processed to serum, and stored at−80° C. until analysis. Study durations were 28 or 49 days. Animals werenot harmed during these studies.

Antibody concentrations were determined using two ELISA assays. In thefirst two studies (referred to as Study 1 and Study 2), goat anti-humanFc antibody (Jackson Immuno research) was adhered to the plate, wellswere washed with PBST (phosphate buffered saline with 0.05% Tween) andblocked with 3% BSA in PBST. Serum or calibration standards were theadded, followed by PBST washing, addition of europium labeled anti-humanIgG (Perkin Elmer), and further PBST washing. The time resolvedfluorescence signal was collected. For Studies 3-5, serum concentrationwas detected using a similar ELISA, but recombinant VEGF (VEGF-165,PeproTech, Rocky Hill, N.J.) was used as capture reagent and detectionwas carried out with biotinylated anti-human kappa antibody andeuropium-labeled streptavidin. PK parameters were determined forindividual mice with a non-compartmental model using WinNonLin(Pharsight Inc, Mountain View Calif.). Nominal times and dose were usedwith uniform weighing of points. The time points used (lambda Z ranges)were from 4 days to the end of the study, although all time points wereused for the faster clearing mutants, P257N and P257L.

Five antibody PK studies in mFcRn^(−/−) hFcRn⁺ mice were carried out.FIG. 13 shows serum concentration data for WT and variant IgG1 (Study 3)and IgG2 (Study 5) antibodies respectively. Fitted PK parameters fromall in vivo PK studies carried out in mFcRn^(−/−) hFcRn⁺ mice areprovided in FIG. 14. PK data include half-life, which represents thebeta phase that characterizes elimination of antibody from serum, Cmax,which represents the maximal observed serum concentration, AUC, whichrepresents the area under the concentration time curve, and clearance,which represents the clearance of antibody from serum. Also provided foreach variant is the calculated fold improvement or reduction inhalf-life relative to the IgG1 or IgG2 parent antibody [Foldhalf-life=half-life(variant)/half-life (WT)].

The data show that a number of the engineered Fc variant antibodies withenhanced FcRn affinity at pH 6.0 extend half-life in vivo. FIG. 15 ashows a plot of the in vivo half-life versus the fold FcRn binding forthe IgG1 antibodies, with select variants labeled. Results from repeatexperiments (circled in the figure) indicate that data from the in vivomodel are reproducible. The best single variants include 308F and 434S,the best double variants include 259I/308F, 308F/428L, 308F/434S, and428L/434S, and the best triple variant is 259I/308F/428L. There is ageneral correlation between affinity for FcRn and in vivo half-life, butit is not completely predictive. Notably, variants 257L and 257N, whichimproved FcRn binding by 3.4 and 3.5-fold respectively, reduced in vivohalf-life by 0.6 and 0.3 respectively. The plot also highlights againthe importance of the amino acid identity of substitution at a givenposition—whereas 308F/434S provided substantial half-life improvement,308F/434M was barely better than WT IgG1.

FIG. 15 b shows a plot of the in vivo half-life versus fold FcRn bindingfor the IgG2 variant antibodies with the variants labeled. When the IgG2in vivo data were compared with the IgG1 in vivo data (FIG. 15 c), asurprising result was observed. The variants provided a substantiallygreater improvement to in vivo half-life in the context of an IgG2 Fcregion than they do an IgG1 Fc region. The longest single variant anddouble variant half-lives from all antibodies in all 5 studies were 12.2and 16.5, provided by 434S IgG2 and 428/L434S IgG2 respectively. Thedramatic improvement in half-lives for the IgG2 variants relative toIgG1 were despite the fact that fold-improvements by the variants inIgG2 were comparable or even lower than they were in IgG1 (434S IgG1fold=3.8, 434S IgG2 fold=4.9, 428L/434S IgG1 fold=17.3, 428L/434S IgG2fold=14.8). Thus unexpectedly, the IgG2 antibody may be the bestapplication for the Fc variants for improving in vivo half-life inmammals.

Example 6 Variant Immunoadhesins

The Fc variants of the invention were also evaluated for their capacityto improve the half-life of immunoadhesins (also referred to as Fcfusions). Select Fc variants were engineered into the anti-TNFimmunoadhesion etanercept (Enbrel®). Etanercept is a fusion of human TNFreceptor 2 (TNF RII and the Fc region of human IgG1, and is clinicallyapproved for the treatment of rheumatoid arthritis, juvenile idiopathicarthritis, ankylosing spondylitis, psoriatic arthritis, and psoriasis.An IgG2 Fc region version of this Fc fusion was also constructed, andselect Fc variants were constructed in this context as well. The aminoacid sequences of the anti-TNF immunoadhesins characterized in theinvention are provided in FIG. 16. Genes were constructed usingrecursive PCR and subcloned into the pTT5 vector, and Fc variants wereconstructed using QuikChange® mutagenesis methods. Immunoadhesins wereexpressed in 293E cells and purified as described above.

The binding specificity of the purified immunoadhesins was confirmed bytesting binding to recombinant TNF by Biacore. Immunoadhesins werecaptured onto an immobilized Protein A/G (Pierce) CM5 biosensor chip(Biacore), generated using standard primary amine coupling.Immunoadhesins were immobilized on the Protein AG surface, andrecombinant TNF in serial dilutions was injected over antibody boundsurface, followed by a dissociation phase. After each cycle, the surfacewas regenerated with buffer. Data were processed by zeroing time andresponse before the injection of receptor and by subtracting from areference channel to account for changes due to injections. Kinetic datawere fit to a 1:1 binding model (Langmuir). Equilibrium associationconstants (Ka's) obtained from these fits are provided in FIG. 17. Theresults show that the variant immunoadhesins retained affinity for TNF,comparable to commercial enbrel.

Variant immunoadhesins were tested for binding to human FcRn at pH 6.0using Biacore as described above. The results (FIG. 18) indicate that,similar as in the context of antibodies, the variants improve binding toFcRn relative to their IgG1 and IgG2 parent immunoadhesin proteins.

The half-lives of the variant immunoadhesins were tested in themFcRn^(−/−) hFcRn⁺ mice as described above. 12 mice per group wereinjected at 2 mg/kg of variant and parent IgG1 immunoadhesin. Serumconcentration was detected using an ELISA similar to that describedabove, except that goat anti-human TNF RII antibody was used as capturereagent; detection was carried out with biotinylated anti-human kappaantibody and europium-labeled streptavidin. FIG. 19 shows serumconcentration data for WT IgG1 Fc and variant Fc immunoadhesins. FittedPK parameters, as described above, from the PK study are provided inFIG. 20. Also provided for each variant is the calculated % increase inhalf-life, calculated as 100 times the half-life of variant Fc fusionover that of the WT IgG1 Fc parent. The results indicate that thevariants extend in vivo half-life in the context of the immunoadhesin.

Example 7 Pharmacokinetic Experiment in Nonhuman Primates

The PK properties of biologics in non-human primates arewell-established to be predictive of their properties in humans. A PKstudy was carried out in cynomolgus monkeys (macaca fascicularis) inorder to evaluate the capacity of the variant anti-VEGF antibodies toimprove serum half-life in non-human primates.

In preparation for a PK study in cynomolgus monkeys, binding of thevariant antibodies to cynomolgus (cyno) FcRn (cFcRn) at pH 6.0 wasmeasured. cFcRn was constructed, expressed, and purified as describedabove for human FcRn. Binding of variant anti-VEGF antibodies to cFcRnwas measured using Biacore as described above. The data are provided inFIG. 21. The results show that the variants improve affinity for cynoFcRn similarly as they do for human FcRn. Dissociation at the higher pH(7.4) was also very rapid (data not shown), similar to as was observedfor binding to human FcRn. These results are not surprising given thehigh sequence homology of the human and cyno receptors (FcRn alpha chain96%, beta-2-microglobulin 91%).

The PK of the variants were studied in vivo in non-human primates. Malecynomolgus monkeys (macaca fascicularis, also called crab-eatingMacaque) weighing 2.3-5.1 kg were randomized by weight and divided into5 groups with 3 monkeys per group. The monkeys were given a single, 1hour peripheral vein infusion of 4 mg/kg antibody. Blood samples (1 ml)were drawn from a separate vein from 5 minutes to 90 days aftercompletion of the infusion, processed to serum and stored at −70 C.Animals were not harmed during these studies.

Antibody concentrations were determined using the VEGF capture method asdescribed above. PK parameters were determined by fitting theconcentrations versus time to a non-compartmental model as was done inthe mouse PK studies. However, time points from day 10 to day 90 wereused for PK parameter determinations. The PK results are plotted in FIG.22, and the fitted parameters are provided in FIG. 23. The results showthat the variants enhanced the in vivo half-life of antibody up to3.2-fold. In the best case (the 428L/434S variant) half-life wasextended from 9.7 days to 31.1 days. The PK results obtained incynomolgus monkeys are consistent with those obtained in mFcRn^(−/−)hFcRn⁺ mice, validating the hFcRn mouse model as a system for assessingthe in vivo PK properties of the variants, and supporting theconclusions from those studies.

Whereas particular embodiments of the invention have been describedabove for purposes of illustration, it will be appreciated by thoseskilled in the art that numerous variations of the details may be madewithout departing from the invention as described in the appendedclaims. All references cited herein are incorporated in their entirety.

1. A method of increasing antibody serum half-life in a patientcomprising administering to said patient an antibody comprising avariant Fc domain as compared to a parent Fc polypeptide, wherein saidvariant Fc domain comprises a leucine at position 428 and a serine atposition 434 in the Fc region of said antibody, wherein said antibodyhas increased in vivo half-life as compared to an antibody comprisingsaid parent Fc polypeptide, and wherein numbering is according to the EUindex in Kabat et al.